Modified ghrelin peptides

ABSTRACT

The present invention provides a novel peptide-type compound which induces secretion of growth hormone and which has the activity of increasing the intracellular calcium ion concentration, wherein at least one amino acid is replaced by a modified amino acid and/or a non-amino acid compound, or a pharmaceutically acceptable salt thereof.

This is a divisional of application Ser. No. 09/959,577, filed Oct. 30,2001, now allowed, which is a 371 National Stage application ofPCT/JP00/04907, filed Jul. 24, 2000. The above-identified applicationsare incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a novel peptide having the action ofincreasing the intracellular calcium concentration or the activity ofinducing secretion of growth hormone, wherein an amino acid in thepeptide is modified. Further, the present invention relates to a methodfor obtaining said novel peptide and a method for producing the same, agene coding said peptide or a precursor of said peptide, and a methodfor producing said peptide or a precursor of said peptide by use of saidgene. Further, the present invention relates to a structural analogue ofthe novel modified peptide disclosed in the present invention, whichbinds to a receptor for a growth hormone secretion-inducing compoundthereby exhibiting the action of increasing the intracellular calciumconcentration or the activity, of inducing secretion of growth hormone,as well as a method for producing the same. Further, the presentinvention relates to a pharmaceutical composition or a growth promoterfor animals comprising said peptide or said peptide analogue as anactive ingredient, as well as an antibody to said peptide or a method ofutilizing the same.

BACKGROUND ART

Growth hormone (abbreviated hereinafter to GH) is a proteinous hormonesynthesized in adenohypophysis and indirectly promotes growth of boneand differentiation of adipocytes and chondrocytes, and its secretion ispromoted by growth hormone-releasing hormone (GHRH) and inhibited bysomatostatin [J. Kendrew, et al., Eds., The Encyclopedia of MolecularBiology (Blackwell Science Ltd., London, 1994), p. 462]. GH has not onlya growth-promoting action but also actions such as promotion of proteinsynthesis in various tissues, stimulation of transfer of depot fats andelevation of glycogen content in muscles, and a reduction in GHsecretion induces dwarfism, while excessive secretion thereof inducesgigantism or acromegaly [Iwanami's Dictionary of Biology, fourthedition, edited by Ryuichi Yasugi, et al. (Iwanami Syoten, Tokyo, 1997),p. 757].

Since human GH has been produced by genetic engineering, GH is used notonly for treatment of dwarfism [J. O. Jorgensen, Endocr. Rev. 12, 189(1991)], but also for treatment of other diseases, and its variouseffects were found [J. O. Jorgensen, et al., Horm. Res. 42, 235 (1994)].For example, such effects include activation of reconstitution ofosteoblasts and bone in the normal [K. Brixen, et al., Miner. Res. 5,609 (1990)], enhancement of muscular strength and muscular amount inGH-deficient adults [R. C. Cuneo, et al., J. Appl. Physiol. 70, 688(1991)], improvement of motility in GH-deficient adults [R. C. Cuneo, etal., J. Appl. Physiol. 70, 695 (1991)], remedy of heavy burns inchildren [D. N. Herndon, et al., Ann. Surg. 212, 424 (1990)], itscombined use with gonadotropins in induction of ovulation [R. Homburg,et al., Clin. Endocrinol. (Oxf). 32, 781 (1990)], prevention ofmetabolic disorder by administration of prednisone [F. F. Horber and M.W. Haymond, J. Clin. Invest. 86, 265 (1990)], promotion of T cell“education” in heavy immune disorder [W. J. Murphy, et al., Proc. Natl.Acad. Sci. U.S. A. 89, 4481 (1992)], and the effect of inhibitingreduction of the body weight of the aged and the effect of enlargingadipose fat tissues and preventing dermal atrophy [D. Rudman, et al, N.Engl. J. Med. 323, 1 (1990)].

Administration of recombinant GH is effective for promotion of growth inchildren and normalization of defects in metabolism and functionsaccompanying GH-deficiency in adults, but there are problems that GH hasdose-restricting side effects, cannot be orally administered and isexpensive [B. A. Lefker, et al., in Growth Hormone Secretagogues inClinical Practice, B. B. Bercu and R. F. Walker, Eds. (Marcel Dekker,Inc., New York, 1998), pp. 107-108]. Many adult patients suffer fromside effects such as arthralgia and a carpal tunnel syndrome consideredto be attributable to pool of excess sodium and humor, so that GHadministration cannot be continued [E. Corpas, et al., Endocr. Rev. 14,20 (1993)]. These side effects are correlated with a non-physiologicalpattern of hormone secretion by GH administration, and in GHadministration, the pulsatility of normal GH secretion cannot beimitated [B. A. Lefker, et al., in Growth Hormone Secretagogues inClinical Practoce, B. B. Bercu and R. F. Walker, Eds. (Marcel Dekker,Inc., New York, 1998), pp. 107-108].

The pulsatility of in vivo GH secretion is established basically byinteraction between two regulating factors derived from hypothalamus;that is, GHRH and somatostatin act on pituitary gland to regulate GHsecretion [G. S. Tannenbaum and N. Ling, Endocrinology 115, 1952 (1984),R. G. Clark and I. C. Robinson, Endocrinology 122, 2675 (1988)]. Thenormal pattern of GH secretion differs during the day and night, andduring the night, a larger amount of GH is released more frequently. Theamplitude of GH release pulse is further regulated by feedback byvarious steroid hormones, neurotransmitters, GH and insulin-like growthfactor, by nutritional status, sleep and motility [J. S. Strobl and M.J. Thomas, Pharmacol. Rev. 46, 1 (1994)].

To overcome the side effects caused by GH administration, a large numberof compounds having a GH secretion-inducing action were synthesized, andas growth hormone secretagogue (GHS), their structural activitycorrelation, their pharmacology and clinical applications wereextensively studied. First, peptides such as GHRP-6 (GrowthHormone-Releasing hexapeptide) were synthesized and developed astherapeutic agents for treating disorders attributable to deficiency orreduction in GH [C. Y. Bowers, et al., Endocrinology 114, 1537-1545(1984)]. However, because these peptide compounds could demonstratetheir effect through intravenous injection only, non-peptide compoundshaving low-molecular weight capable of oral administration weredeveloped [R. G. Smith, et al., Science 260, 1640-1643 (1993)] and someof them have advanced to a phase II clinical test [A. A. Patchett, etal., Proc. Natl. Acad. Sci. U.S.A. 92, 7001-7005 (1995)].

A series of information transfer from signal reception of receptor tofunctional expression is called signal transduction, and the signaltransduction system coupled with G protein proceeds in the followingmechanism [Iwanami's Dictionary of Biology, fourth edition, ed. byRyuichi Yasugi, et al., pp. 555-556 (Iwanami Syoten, Tokyo, 1997)]. ThisG protein coupled system has a receptor with seven transmembrane domainsand is divided into a cAMP system for producing cAMP as a secondmessenger and inositol-1,4,5-triphosphoric acid (IP3) and diacylglycerol(DG) inositol phospholipid information transduction system. The cAMPactivates cAMP-dependent kinase (A kinase), to cause phosphorylation ofserine and threonine residues in functional protein to modify itsactivity. On the other hand, IP3 binds to IP3 receptor on endoplasmicreticulum to promote release of calcium ions, while DG activates Ckinase to promote the action of hormones etc.

The mechanism of increasing the intracellular calcium ion concentrationin the signal transduction system with IP3 or DG as second messenger [J.Kendrew, et al., Eds., The Encyclopedia of Molecular Biology (BlackwellScience Ltd., London, 1994), p. 136-137] is as follows: When a ligandbinds to the receptor, phospholipase C is activated via G protein, tocovert PIP2 into IP3. By IP3, calcium ions pooled in endoplasmicreticulum (ER) as intracellular granule are released into cytoplasm,thus increasing calcium ion levels in the cytoplasm. If IP3 or calciumions are present in the cytoplasm, the calcium is incorporated againinto the endoplasmic reticulum, thus lowering calcium ion levels in thecytoplasm. That is, the binding of the ligand to the receptor causes atransient increase in calcium ion levels in the cytoplasm.

Since GHS acts synergistically on the GH secretion and increase ofintracellular cAMP levels by GHRH [K. Cheng, et al., Endocrinology 124,2791-2798 (1989)] and the binding of GHRH to the receptor inducesproduction of cAMP as second messenger while GHS induces an increase inthe intracellular calcium ion concentration, it was suggested that theworking mechanism of GHS is different from that of GHRH [J. Herringtonand B. Hille, Endocrinology 135, 1100-1108 (1994).], and GHS wassupposed to bind to a receptor different than GHRH receptor. Actually, agene for a receptor to which GHS is bound was cloned, and from theresult of Northern analysis, it was found that GHS receptor (GHS-R) isexpressed in hypothalamus and brain pituitary gland, and that there is90% or more homology between the amino acid sequences of porcine- andhuman-derived GHS receptors [A. D. Howard, et al., Science 273, 974-977(1996)]. However, an endogenous ligand that binds to GHS-R has not beenisolated, and this GHS-R was an orphan receptor whose ligand was notevident.

In some cases, fatty acids such as myristic acid, geranic acid,palmitoyl acid or farnesyl acid are bound to the amino-terminal of acertain protein or to side chains of its amino acid residues, and therole of these fatty acids is anchoring such fatty acid-modified proteinto cell membrane [J. Kendrew, et al., Eds., The Encyclopedia ofMolecular Biology (Blackwell Science Ltd., London, 1994), p. 616]. Insuch fatty acid-modified protein, the fatty acid binds to a cysteineresidue via S-acyl linkage, and neither an amino acid having fatty acidbound to serine residue via O-acyl linkage, such as the endogenous GHSdisclosed in the present invention, nor protein or peptide containingsuch fatty acid-modified amino acid, is known. Neither is it known forwhich the peptide containing such fatty acid-modified amino acidfunctions as a ligand for any receptor.

DISCLOSURE OF INVENTION

Before the present invention is described in detail, terms are definedas follows:

The term “peptide” refers to a compound comprising a plurality of aminoacids linked therein via peptide linkages. Here, the amino acid (alsocalled an amino acid residue) includes naturally occurring amino acidsrepresented by formula: NH₂—CH(R′)—COOH, wherein R′ is a naturallyoccurring substituent group, as well as its D, L-optical isomers etc.

There is also a peptide, wherein a certain naturally occurring aminoacid is replaced by a modified amino acid (also called a modified aminoacid residue). The modified amino acid includes the amino acids of theabove formula wherein the substituent group R′ is further modified, itsD, L-optical isomers thereof, and non-natural amino acids wherein e.g.various substituent groups are bound to the substituent group R′ of theabove formula via or not via an ester, ether, thioester, thioether,amide, carbamide or thiocarbamide linkage. The modified amino acid alsoincludes non-natural amino acids whose amino groups are replaced bylower alkyl groups.

The terms “peptide analogue” refer to a compound wherein at least oneamino acid in a peptide is replaced by a non-amino acid compound, andthus at least one linkage of said substituent compound to the peptideanalogue is not a peptide linkage.

Further, those compounds derived from these peptides and peptideanalogues by modifying the amino-terminal and/or carboxyl-terminalthereof are referred to as derivatives. And the peptides, peptideanalogues and derivatives thereof are referred collectively to as“peptide-type compound”.

In the amino acid sequence set forth in SEQ ID NO: 2, an amino acidsequence of amino acids 1 to 4 refers to Gly Ser Ser Phe,

an amino acid sequence of amino acids 1 to 5 refers to Gly Ser Ser PheLeu,an amino acid sequence of amino acids 1 to 6 refers to Gly Ser Ser PheLeu Ser,an amino acid sequence of amino acids 1 to 7 refers to Gly Ser Ser PheLeu Ser Pro,an amino acid sequence of amino acids 1 to 8 refers to Gly Ser Ser PheLeu Ser Pro Glu,an amino acid sequence of amino acids 1 to 9 refers to Gly Ser Ser PheLeu Ser Pro Glu His, andan amino acid sequence of amino acids 1 to 10 refers to Gly Ser Ser PheLeu Ser Pro Glu His Gln.

The discovery of an endogenous ligand (endogenous GHS) which binds toGHS receptor to exhibit an activity for increasing the intracellularcalcium ion concentration or for inducing GH secretion has been desiredtogether with a method of utilizing the same. Further, a compound hasbeen desired, which is a structural analogue of said endogenous GHS andhas an activity for increasing the intracellular calcium ionconcentration or for inducing GH secretion. Further, a pharmaceuticalcomposition or a composition for promoting animal growth has beendesired, which comprises said endogenous GHS or its structural analogousinducing pulsatile GH secretion thereby eliminating side effects by GHadministration, as well as a therapeutic application using saidcomposition.

The present inventors focused their attention on the fact that bindingof the ligand to GHS receptor (GHS-R) causes a transient increase in theintracellular calcium ion concentration with inositol phospholipid assecond messenger, and they screened extracts of various organs andtissues by using the activity of increasing the intracellular calciumion concentration (Ca-releasing activity) as an indicator in CHO cells(CHO-GHSR62) expressing GHS-R. As a result, the inventors found thatstomach extracts of rat has a strong Ca-releasing activity, andsuccessfully purified a substance having a strong Ca-releasing activityfrom the above extracts by various kinds of chromatography, and foundthat said substance is a novel peptide modified with fatty acid, havinga molecular weight of about 3,000. Further, they confirmed that saidnovel peptide promotes specific secretion of GH from cells of anteriorpituitary, and found that said novel peptide is an endogenous ligand forGHS-R, that is, an endogenous GH secretagogue (endogenous GHS). That is,the first aspect of the present invention is directed to an endogenousGH secretion-inducing peptide having the activity of increasing theintracellular calcium ion concentration or the activity of inducing GHsecretion, wherein a certain constituent amino acid residue is modifiedwith fatty acid, as well as a method for preparing said peptide.

The present inventors precisely analyzed the structure of the endogenousGH secretion-inducing peptide, and found that said peptide is a peptideconsisting of the amino acid sequence set forth in SEQ ID NO: 2, whereinthe side-chain hydroxyl group of 3rd serine from the Amino-terminal hasbeen acylated with fatty acid. Further, a human-derived GHsecretion-inducing peptide was also purified from human stomach extracthaving a strong Ca-releasing activity similar to that of rat stomachextract and analyzed for its structure in the same manner as forrat-derived GH secretion-inducing peptide, and as a result the inventorsfound that the human-derived endogenous GH secretion-inducing peptideconsists of the amino acid sequence set forth in SEQ ID NO: 3, whereinthe side-chain hydroxyl group of 3rd serine from the amino-terminal hasbeen acylated with fatty acid. Comparison between the amino acidsequences of the rat- and human-derived endogenous GH secretion-inducingpeptides revealed homology as high as 89% as a whole.

Specifically, the rat- and human-derived peptides are identical in anamino acid sequence of amino acids 1 to 10 from amino-terminal and in anamino acid sequence of amino acids 13 to 28 from amino-terminal, but aredifferent in amino acids 11 and 12 which are lysine and alanine in therat peptide, which are replaced by arginine and valine in the humanpeptide, respectively. The rat-derived endogenous GH secretion-inducingpeptide was cleaved with various proteases and its purified peptidefragments were measured for Ca-releasing activity, and as a result, apeptide consisting of amino acids 1 to 7 from amino-terminal was theminimum peptide having the Ca-releasing activity.

By measurement of the Ca-releasing activity of chemically synthesizedpeptides, the inventors found that the core sequence essential foreliciting the Ca-releasing activity is a sequence consisting of 4 aminoacids set forth in SEQ ID NO: 8. Further, the sequence consisting of 10amino acids set forth in SEQ ID NO: 9 was conserved in non-ratendogenous GH secretion-inducing peptides (each consisting of 28 aminoacids) separated from human, porcine and bovine, as well as inendogenous GH secretion-inducing peptides (each consisting of 27 aminoacids) wherein one glutamine was deleted from the above peptides.

That is, the second aspect of the present invention is directed to afatty acid-modified peptide comprising the amino acid sequence set forthin SEQ ID NO: 8, preferably the amino acid sequence set forth in SEQ IDNO: 1 and more preferably the amino acid sequence set forth in SEQ IDNO: 9 as the core sequence essential for eliciting the Ca-releasingactivity.

Endogenous GH secretion-inducing peptides were also isolated fromchicken, eel and frog, and these peptides were found to have a coresequence consisting of 4 amino acids set forth in SEQ ID NO: 8.

In addition, an endogenous GH secretion-inducing peptide very similar tothe rat endogenous GH secretion-inducing peptide was also isolated fromfrog.

Further, endogenous GH secretion-inducing peptides were also isolatedfrom Xenopus laevis, rainbow trout (Oncorhynchus mykiss), and dog. Fromrainbow trout, ghrelin-23 consisting of 23 amino acids and ghrelin-20consisting of 20 amino acids were isolated respectively.

The carboxyl-terminal amino acid of eel ghrelin, rainbow troutghrelin-23 and ghrelin-20 was amidated.

Because an amino acid residue at the 3rd position from theamino-terminal in the endogenous GH secretion-inducing peptide fromXenopus laevis is threonine, the present invention also relates to afatty acid-modified peptide, which contains, as the core sequenceessential for exhibiting the Ca-releasing activity, a peptide whereinthe amino acid residue 3rd serine was replaced by threonine in the aminoacid sequence set forth in SEQ ID NO: 8, preferably the amino acidsequence set forth in SEQ ID NO: 1 and more preferably the amino acidsequence set forth in SEQ ID NO: 9.

The endogenous fatty acid-modified peptide having GH secretion-inducingactivity or the fatty acid-modified peptide consisting of said coresequence, disclosed in the present invention, also provides a guidelinefor designing a compound having Ca-releasing activity.

That is, in the third aspect of the present invention, a novel compoundhaving Ca-releasing activity is obtained by synthesizing a structuralanalogue of said fatty acid-modified peptide by confirming theCa-releasing activity of the resulting structural analogue. Accordingly,the present invention also encompasses a peptide or peptide analoguehaving the activity of increasing the intracellular calcium ionconcentration, wherein a certain constituent amino acid is replaced by amodified amino acid or non-amino acid compound.

A cDNA coding the endogenous GH secretion-inducing peptide was obtainedin a usual manner. Each of rat and human cDNAs consists of 117 aminoacids as shown in the amino acid sequences in SEQ ID NOS: 4 and 5, andthe amino acid sequences of rat and human endogenous GHsecretion-inducing peptides were identical in a sequence of 28 aminoacids from the 24th to 51st positions from the amino-terminal,respectively. That is, it was revealed that the endogenous GHsecretion-inducing peptide is synthesized as a precursor peptideconsisting of 117 amino acids, then a signal peptide consisting ofamino-terminal 23 amino acids is cleaved off and furthercarboxyl-terminal 56 amino acids are cleaved off, whereby the fattyacid-modified peptide having GH secretion-inducing activity is formed.In addition, a cDNA coding a precursor of the endogenous GHsecretion-inducing peptide consisting of 28 amino acids was also foundin porcine.

Further, a cDNA coding a precursor for the endogenous GHsecretion-inducing peptide consisting of 27 amino acids was found inporcine.

Further, a partial cDNA coding a precursor for the endogenous GHsecretion-inducing peptide consisting of 27 amino acids was found inbovine.

Further, a cDNA coding a precursor of the endogenous GHsecretion-inducing peptide was also found in eel, Xenopus laevis andrainbow trout. From rainbow trout, a cDNA coding a precursor ofghrelin-23 consisting of 23 amino acids and a cDNA coding a precursor ofghrelin-20 of 20 amino acids were isolated respectively.

Accordingly, the fourth aspect of the present invention lies in a cDNAcoding a precursor of the endogenous GH secretion-inducing peptide, aswell as a method for producing a peptide as a starting material of thefatty acid-modified peptide or peptide analogue having Ca-releasingactivity, which comprises using said cDNA.

In purification of the endogenous GH secretion-inducing peptide(ghrelin) composed of 28 amino acids from rat stomach extract, a peptiderecovered as a minor fraction was analyzed, and as a result a peptideconsisting of 27 amino acids (ghrelin-27), which is a peptide of ghrelinfrom which glutamine 13 or 14 had been deleted, was found. Ghrelin-27has completely the same Ca-releasing activity and GH secretion-inducingactivity as those of ghrelin consisting of 28 amino acids, andghrelin-27 is an endogenous GH secretion-inducing peptide, and thusghrelin-27 also falls under the scope of the present invention.

The nucleotide sequence coding glutamine residues 13 and 14 in ghrelinis gca gca, which is a terminal exon sequence to be subjected to mRNAsplicing, thus suggesting the possibility of formation of a cDNA fromwhich one of two codons for glutamine residues was deleted by differentsplicing. Actually, a cDNA coding a precursor peptide of ghrelin-27consisting of 27 amino acids was found in screening of rat and humancDNA libraries.

That is, it was revealed that rat and human ghrelin-27 peptide issynthesized as a precursor peptide consisting of 116 amino acids setforth in SEQ ID NO: 12 or 13, then a signal peptide consisting ofamino-terminal 23 amino acids is cleaved off and furthercarboxyl-terminal 56 amino acids are cleaved off, whereby a fattyacid-modified peptide consisting of 27 amino acids having GHsecretion-inducing activity (ghrelin-27) is formed.

Further, a cDNA coding a precursor of ghrelin-27 peptide was found inporcine and bovine, and the presence of ghrelin-27 and its precursor wasconfirmed in these animals.

That is, the present invention also encompasses ghrelin-27 peptideconsisting of an amino acid sequence set forth in SEQ ID NOS: 10, 11, 17and 22, a ghrelin-27 precursor peptide having an amino acid sequence setforth in SEQ ID NOS: 12, 13, 19 and 23, and a cDNA coding said precursorpeptide which comprises a nucleotide sequence set forth in SEQ ID NOS:14, 15, 21 and 24.

The fatty acid-modified peptide having Ca-releasing activity and thepeptide-type compound such as peptide analogue having Ca-releasingactivity as disclosed in the present invention also provide apharmaceutical composition for treating diseases attributable to defector decrease in GH. Said pharmaceutical composition can be used to treatany diseases against which the GH administration is effective, andvarious side effects caused by GH administration can be overcome.Further, said pharmaceutical composition can also be used as an animaldrug such as a growth-promoting agent for animals.

Because the peptide-type compound of the present invention has anappetite-promoting action by administration into ventricle andintravenous administration, and thus it can be used as an appetitepromoting agent for treating loss of appetite or sitophobia. Inaddition, the present peptide-type compound has a stomach motility- andgastric acid secretion-promoting action, and thus it can also be used asan agent for treating stomach functional diseases such as non-ulcerindigestion, sudden light stomach atony, functional indigestion, andreflux esophagitis. Further, the present peptide-type compound exhibitsa cell growth-promoting action in bone marrow, duodenum and jejunum byintravenous administration, and thus it can be used as an agent forprotecting mucous membrane on intestinal tract, an agent for preventingdamage to mucous membrane on small intestine during intravenousnutrition and an agent for treating osteoporosis.

The present invention also encompasses an antibody prepared by using thefatty acid-modified peptide having Ca-releasing activity disclosed inthe present invention as an antigen, a method for measuring theendogenous GH secretion-inducing peptide by use of said antibody, and ameasurement kit comprising said antibody.

Further, the present invention encompasses an assay method forseparating and quantifying ghrelin modified with a fatty acid andghrelin from which the fatty acid was eliminated, which comprises usingtwo antibodies built up to N- and carboxyl-terminal peptides fromghrelin, the former antibody capable of recognizing fatty acid-modified3rd serine, as well as an assay kit comprising a combination of theantibodies against N- and carboxyl-terminal peptides from ghrelin.

That is, the present invention provides a novel peptide hormone having anovel modified amino acid i.e. acylated serine, and also provides aguideline for novel design of a compound having Ca-releasing activitywith the structure of said peptide as a fundamental skeleton.

Further, the elucidation of the mechanism of induction of GH secretionby the fatty acid-modified peptide disclosed in the present invention orby GH releasing hormone and somatostatin is suggested to be extendablenot only to the mechanism of induction of GH secretion but also to themechanism of regulating secretion of other hormones. The presentinvention discloses various functions of the fatty acid-modified peptideas a regulatory factor in the circulatory system and the metabolicsystem, and the effect of the present invention extends to theelucidation of a new biological regulatory mechanism.

Specifically, the present invention relates to:

(1) A peptide-type compound, wherein in a peptide having the activity ofincreasing the intracellular calcium ion concentration, at least oneamino acid is replaced by a modified amino acid and/or a non-amino acidcompound, or a pharmaceutically acceptable salt thereof;

(2) The peptide-type compound according to item (1), which comprises (a)an amino acid sequence set forth in SEQ ID NO: 2 or (b) an amino acidsequence having any one of amino acid sequences selected from the groupconsisting of

(1) amino acid sequence of amino acids 1 to 4,(2) amino acid sequence of amino acids 1 to 5,(3) amino acid sequence of amino acids 1 to 6,(4) amino acid sequence of amino acids 1 to 7,(5) amino acid sequence of amino acids 1 to 8,(6) amino acid sequence of amino acids 1 to 9, and(7) amino acid sequence of amino acids 1 to 10from the amino-terminal in the sequence (a) and at least one amino aciddeleted, replaced and/or added in a part outside said amino acidsequences, or a pharmaceutically acceptable salt thereof;

(3) The peptide-type compound according to item (2) above, whichcomprises one amino acid sequence selected from the group consisting ofamino acid sequences set forth in SEQ ID NOS: 3, 4, 5, 8, 9, 10, 11, 12,13, 16, 17, 18, 19, 22 and 23, or a pharmaceutically acceptable saltthereof;

(4) The peptide-type compound according to item (2) above, whichcomprises one amino acid sequence selected from the group consisting ofamino acid sequences set forth in SEQ ID NOS: 25, 26, 29, 30, 31, 32, 34and 35, or a pharmaceutically acceptable salt thereof;

(5) A peptide-type compound, wherein in a peptide having the activity ofincreasing the intracellular calcium ion concentration and the activityof inducing secretion of growth hormone, (a) constitutional amino acidsare modified or not modified and (b) at least one amino acid is replacedor not replaced by a non-amino acid compound, or a pharmaceuticallyacceptable salt thereof;

(6) The peptide-type compound according to item (1) or (5) whichcomprises amino acid sequences set forth in SEQ ID NOS: 27, 28 and 33,or a pharmaceutically acceptable salt thereof;

(7) The peptide-type compound according to item (5), which comprises (a)an amino acid sequence set forth in SEQ ID NO: 2 or (b) an amino acidsequence having any one of amino acid sequences selected from the groupconsisting of

(1) amino acid sequence of amino acids 1 to 4,(2) amino acid sequence of amino acids 1 to 5,(3) amino acid sequence of amino acids 1 to 6,(4) amino acid sequence of amino acids 1 to 7,(5) amino acid sequence of amino acids 1 to 8,(6) amino acid sequence of amino acids 1 to 9, and(7) amino acid sequence of amino acids 1 to 10from the amino-terminal in the sequence (a) and at least one amino aciddeleted, replaced and/or added in a part outside said amino acidsequences, or a pharmaceutically acceptable salt thereof;

(8) The peptide-type compound according to item (7) above, whichcomprises one amino acid sequence selected from the group consisting ofamino acid sequences set forth in SEQ ID NOS: 3, 4, 5, 8, 9, 10, 11, 12,13, 16, 17, 18, 19, 22 and 23, or a pharmaceutically acceptable saltthereof;

(9) The peptide-type compound according to item (7) above, whichcomprises one amino acid sequence selected from the group consisting ofamino acid sequences set forth in SEQ ID NOS: 25, 26, 29, 30, 31, 32, 34and 35, or a pharmaceutically acceptable salt thereof;

(10) The peptide-type compound according to item (1) or (5) above, whoseamino-terminal amino acids 1 to 4 are represented by formula:

A-B-C-D-

wherein the symbol A is either an amino acid or a non-amino acidcompound, or is missing, and the symbol B is either an amino acid or anon-amino acid compound, or is missing, provided that the length of theA+B molecular chain is a dipeptide length, and the symbol C or thesymbol D may be the same or different and represents (a) a modifiedamino acid, (b) an amino acid having a hydrophobic residue, or (c) anamino acid having a basic side chain, or a pharmaceutically acceptablesalt thereof;

(11) The peptide-type compound according to item (10), wherein thesymbol C is a modified amino acid in which (a) a saturated orunsaturated alkyl chain containing one or more carbon atoms wasintroduced at the α carbon atom of the amino acid via or not via analkylene group containing one or more carbon atoms and via an ester,ether, thioether, amide or disulfide linkage, or (b) a saturated orunsaturated alkyl chain containing one or more carbon atoms wasintroduced at the α carbon atom of the amino acid, and the symbol D isan amino acid having a hydrophobic residue, or a pharmaceuticallyacceptable salt thereof;

(12) A peptide-type compound, wherein in one amino acid sequenceselected from the group consisting of amino acid sequences set forth inSEQ ID NOS: 2, 3, 9, 10, 11, 16, 17, 22, 25, 26, 27, 28, 29, 30 and 31,an amino acid sequence of amino-terminal amino acids 1 to 4 is replacedby the structure of the peptide-type compound described in item (10) or(11), or a pharmaceutically acceptable salt thereof;

(13) The peptide-type compound according to item (1), (2), (3), (5), (7)or (8) above, wherein the modified amino acid is an amino acid at the3rd position from the amino-terminal, or a pharmaceutically acceptablesalt thereof;

(14) The peptide-type compound according to item (13), wherein the aminoacid in the modified amino acid is serine or cysteine, or apharmaceutically acceptable salt thereof;

(15) The peptide-type compound according to item (1), (2), (3), (5), (7)or (8) above, which comprises a modified amino acid in which (a) asaturated or unsaturated alkyl chain containing one or more carbon atomswas introduced at the α carbon atom of the amino acid via or not via analkylene group containing one or more carbon atoms and via an ester,ether, thioester, thioether, amide or carbamide linkage, or (b) H or asaturated or unsaturated alkyl chain containing one or more carbon atomswas introduced at the α carbon atom of the amino acid, or apharmaceutically acceptable salt thereof;

(16) The peptide-type compound according to item (1), (2), (4), (5),(6), (7), (9), (10) or (12) above, wherein the modified amino acid is anamino acid in which, (a) a saturated or unsaturated alkyl chaincontaining one or more carbon atoms was introduced at the α carbon atomvia or not via an alkylene group containing one or more carbon atoms andvia an ester, ether, thioester, thioether, disulfide, amide, carbamideor thiocarbamide linkage, or (b) a saturated or unsaturated alkyl chaincontaining one or more carbon atoms was introduced at the a carbon, or apharmaceutically acceptable salt thereof;

(17) The peptide-type compound according to item (1), (2), (3), (5), (7)or (8) above, which comprises a modified amino acid modified with anester linkage, or a pharmaceutically acceptable salt thereof;

(18) The peptide-type compound according to item (1), (2), (4), (5),(6), (7), (9), (10), (11) or (12) above, which comprises a modifiedamino acid modified by conversion of a functional group in a side chainof said amino acid into an ester linkage, or a pharmaceuticallyacceptable salt thereof;

(19) The peptide-type compound according to item (17) above, whichcomprises an amino acid having a fatty acid bound via an ester linkageto a side-chain hydroxyl group of said amino acid, or a pharmaceuticallyacceptable salt thereof;

(20) The peptide-type compound according to item (18) above, whichcomprises an amino acid having a fatty acid bound via an ester linkageto a side-chain hydroxyl group of said amino acid or via a thioesterlinkage to a side-chain mercapto group of said amino acid, or apharmaceutically acceptable salt thereof;

(21) The peptide-type compound according to item (19) above, whichcomprises an amino acid to which a fatty acid containing 2 to 35 carbonatoms was bound, or a pharmaceutically acceptable salt thereof;

(22) The peptide-type compound according to item (20) above, wherein thefatty acid contains 2 to 35 carbon atoms, or a pharmaceuticallyacceptable salt thereof;

(23) The peptide-type compound according to item (21) above, whichcomprises an amino acid to which a fatty acid selected from the groupconsisting of fatty acids containing 2, 4, 6, 8, 10, 12, 14, 16 and 18carbon atoms was bound, or a pharmaceutically acceptable salt thereof;

(24) The peptide-type compound according to item (22) above, wherein thefatty acid is a fatty acid selected from the group consisting of fattyacids containing 2, 4, 6, 8, 10, 12, 14, 16 and 18 carbon atoms, or apharmaceutically acceptable salt thereof;

(25) The peptide-type compound according to item (23) above, wherein thebound fatty acid is octanoic acid, a monoene fatty acid thereof or apolyene fatty acid thereof, or a pharmaceutically acceptable saltthereof;

(26) The peptide-type compound according to item (24) above, wherein thefatty acid is octanoic acid, a monoene fatty acid thereof or a polyenefatty acid thereof, or a pharmaceutically acceptable salt thereof;

(27) The peptide-type compound according to item (23) above, wherein thebound fatty acid is decanoic acid, a monoene fatty acid thereof or apolyene fatty acid thereof, or a pharmaceutically acceptable saltthereof;

(28) The peptide-type compound according to item (24) above, wherein thefatty acid is decanoic acid, a monoene fatty acid thereof or a polyenefatty acid thereof, or a pharmaceutically acceptable salt thereof;

(29) A peptide-type compound comprising a basic amino acid bound to thecarboxyl-terminal of a peptide-type compound described in items (1) to(28) above;

(30) The peptide-type compound according to items (1), (2), (3), (5),(7), (8), (13), (14), (15), (17), (19), (21), (23), (25) and (27) above,wherein the amino-terminal is modified with a saturated or unsaturatedalkyl or acyl group containing one or more carbon atoms, and/or ahydroxyl group of the carboxyl-terminal carboxyl group is OZ or NR2R3wherein Z is a pharmaceutically acceptable cation or a lower branched orlinear alkyl group, and R2 and R3 are the same or different andrepresent H or a lower branched or linear alkyl group;

(31) The peptide-type compound according to items (1), (2), (4), (5),(6), (7), (9), (10), (11), (12), (16), (18), (20), (22), (24), (26),(28) and (29) above, wherein the amino-terminal amino group is modifiedby introduction of a saturated or unsaturated alkyl or acyl groupcontaining one or more carbon atoms, and/or a hydroxyl group of thecarboxyl-terminal carboxyl group is OZ or NR2R3 wherein Z is apharmaceutically acceptable cation or a lower branched or linear alkylgroup, and R2 and R3 are the same or different and represent H or alower branched or linear alkyl group;

(32) A peptide-type compound comprising a basic group introduced into acarboxyl-terminal amide derivative of a peptide-type compound describedin item (30) or (31) above;

(33) A pharmaceutical composition comprising a peptide-type compounddescribed in items (1) to (32) above or a pharmaceutically acceptablesalt thereof as an active ingredient;

(34) A pharmaceutical composition for treatment of diseases attributableto a defect or decrease in growth hormone, which comprises apeptide-type compound described in items (1) to (32) above or apharmaceutically acceptable salt thereof as an active ingredient;

(35) A pharmaceutical composition for treatment of diseases notattributable to a defect or decrease in growth hormone, which comprisesan agent for treating diseases not attributable to a defect or decreasein growth hormone and a peptide-type compound described in items (1) to(32) above or a pharmaceutically acceptable salt thereof;

(36) A pharmaceutical composition according to items (33) to (35) above,which is applied to animals other than human beings;

(37) A method for treatment of diseases attributable to a defect ordecrease in growth hormone, which comprises administering apharmaceutical composition comprising a peptide-type compound describedin items (1) to (32) above or a pharmaceutically acceptable salt thereofas an active ingredient;

(38) A method for treatment of diseases not attributable to a defect ordecrease in growth hormone, which comprises administering an agent fortreating diseases not attributable to a defect or decrease in growthhormone and a peptide-type compound described in items (1) to (32) aboveor a pharmaceutically acceptable salt thereof;

(39) The treatment method according to items (37) or (38), which isapplied to animals other than human beings;

(40) A DNA coding an amino acid sequence of a peptide-type compounddescribed in items (1) to (32) above, which comprises a nucleotidesequence coding a peptide containing an amino acid sequence recognizingat least one modifiable amino acid in the amino acid sequence encoded bysaid DNA;

(41) The DNA according to item (40) above, wherein the nucleotidesequence is one nucleotide sequence selected from the group consistingof nucleotide sequences set forth in SEQ ID NOS: 6, 7, 14, 15, 20, 21,24, 36, 37, 38 and 39;

(42) The DNA according to item (40) above, wherein the nucleotidesequence is an amino acid-coding nucleotide sequence in one nucleotidesequence selected from the group consisting of nucleotide sequences setforth in SEQ ID NOS: 6, 7, 14, 15, 20, 21, 24, 36, 37, 38 and 39;

(43) A vector comprising a DNA described in items (40) to (42) above;

(44) Cells comprising the vector described in item (43) above;

(45) Cells comprising a DNA described in items (40) to (42) above,wherein a peptide-type compound having an amino acid sequence encoded bysaid DNA can be produced as a peptide-type compound having at least oneamino acid modified in said amino acid sequence;

(46) An antibody against a peptide-type compound described in items (1)to (32) above;

(47) A method for assaying a peptide-type compound described in items(1) to (32) above, which comprises using the antibody described in item(46) above to detect the peptide-type compound described in items (1) to(32) above;

(48) A kit for detecting a peptide-type compound described in items (1)to (32) above, which comprises using the antibody described in item (46)above to detect the peptide-type compound described in items (1) to (32)above;

(49.) A method for producing a peptide-type compound described in items(1) to (32) above by genetic recombination technology, which comprisestransforming a vector containing a DNA described in items (40) to (42)above into host cells capable of modifying a side chain of at least oneamino acid in said peptide, then culturing the resulting transformedcells and recovering the desired peptide-type compound from the culture;

(50) A method for producing a peptide-type compound described in items(1) to (32) above by genetic recombination technology, which comprisestransforming a vector containing a DNA described in items (40) to (42)above into host cells, then culturing the resulting transformed cellsand recovering the desired peptide-type compound from the culture,followed by chemically modifying an arbitrary amino acid thereof;

(51) A method for producing a peptide-type compound described in items(19) to (28) above by genetic recombination technology, which comprisesusing cells having the activity of binding a fatty acid via an esterlinkage to a side-chain hydroxyl group of an amino acid or via athioester linkage to a side-chain mercapto group of an amino acid in thepeptide-type compound;

(52) A method for producing a peptide-type compound described in items(19) to (28) above, which comprises using cells having the serineacylation activity of binding a fatty acid via an ester linkage to aside-chain hydroxyl group of serine in the amino acid sequence set forthin SEQ ID NO: 8;

(53) A process for producing a peptide-type compound described in items(19) to (28) above, which comprises using cells having the acylationactivity of binding a fatty acid via an ester linkage to a side-chainhydroxyl group of threonine in the amino acid sequence set forth in SEQID NO: 28;

(54) A pharmaceutical composition for gene therapy for treatment ofdiseases attributable to a defect or decrease in growth hormone, whichcomprises integrating a vector containing a DNA coding an amino acidsequence of a peptide-type compound described in items (1) to (32) aboveinto cells in a living body and expressing a peptide with at least onemodified amino acid, the peptide having the activity of increasing theintracellular calcium ion concentration;

(55) A method for treatment of diseases attributable to a defect ordecrease in growth hormone, which comprises integrating a vectorcontaining a DNA coding an amino acid sequence of a peptide-typecompound described in items (1) to (32) above into cells in a livingbody enabling a peptide having an amino acid sequence encoded by saidDNA to be produced as a peptide having an amino acid sequencerecognizing at least one modifiable amino acid in said amino acidsequence, whereby a peptide having the activity of inducing growthhormone is expressed;

(56) A pharmaceutical composition for gene therapy for treatment ofdiseases not attributable to a defect or decrease in growth hormone,which comprises integrating a vector containing a DNA coding an aminoacid sequence of a peptide-type compound described in items (1) to (32)above into cells in a living body and expressing a peptide with at leastone modified amino acid, the peptide having the activity of increasingthe intracellular calcium ion concentration;

(57) A method for treatment of diseases not attributable to a defect ordecrease in growth hormone, which comprises integrating a vectorcontaining a DNA coding an amino acid sequence of a peptide-typecompound described in items (1) to (32) above into cells in a livingbody enabling a peptide having an amino acid sequence encoded by saidDNA to be produced as a peptide having an amino acid sequencerecognizing at least one modifiable amino acid in said amino acidsequence, whereby a peptide having the activity of inducing growthhormone is expressed.

Specifically, the present invention also relates to:

(1) A peptide-type compound, wherein in a peptide having the activity ofincreasing the intracellular calcium ion concentration, at least oneamino acid is replaced by a modified amino acid and/or a non-amino acidcompound, or a pharmaceutically acceptable salt thereof;

(2) The peptide-type compound according to item (1) above, whichcomprises (a) an amino acid sequence set forth in SEQ ID NO: 2 or (b) anamino acid sequence having any one of amino acid sequences selected fromthe group consisting of

(1) amino acid sequence of amino acids 1 to 4,(2) amino acid sequence of amino acids 1 to 5,(3) amino acid sequence of amino acids 1 to 6,(4) amino acid sequence of amino acids 1 to 7,(5) amino acid sequence of amino acids 1 to 8,(6) amino acid sequence of amino acids 1 to 9, and(7) amino acid sequence of amino acids 1 to 10from the amino-terminal in the sequence (a) and at least one amino aciddeleted, replaced and/or added in a part outside said amino acidsequences, or a pharmaceutically acceptable salt thereof;

(3) The peptide-type compound according to item (2) above, whichcomprises one amino acid sequence selected from the group consisting ofamino acid sequences set forth in SEQ ID NOS: 3, 4, 5, 8, 9, 10, 11, 12,13, 16, 17, 18, 19, 22, 23, 25 and 26, or a pharmaceutically acceptablesalt thereof;

(4) A peptide-type compound wherein in a peptide having the activity ofincreasing the intracellular calcium ion concentration and the activityof inducing secretion of growth hormone, (a) constitutional amino acidsare modified or not modified and (b) at least one amino acid is replacedor not replaced by a non-amino acid compound, or a pharmaceuticallyacceptable salt thereof;

(5) The peptide-type compound according to item (1) or (4) whichcomprises an amino acid sequence set forth in SEQ ID NO: 27, or apharmaceutically acceptable salt thereof;

(6) The peptide-type compound according to item (4) above, whichcomprises (a) an amino acid sequence set forth in SEQ ID NO: 2 or (b) anamino acid sequence having any one of amino acid sequences selected fromthe group consisting of

(1) amino acid sequence of amino acids 1 to 4,(2) amino acid sequence of amino acids 1 to 5,(3) amino acid sequence of amino acids 1 to 6,(4) amino acid sequence of amino acids 1 to 7,(5) amino acid sequence of amino acids 1 to 8,(6) amino acid sequence of amino acids 1 to 9, and(7) amino acid sequence of amino acids 1 to 10from the amino-terminal in the sequence (a) and at least one amino aciddeleted, replaced and/or added in a part outside said amino acidsequences, or a pharmaceutically acceptable salt thereof;

(7) The peptide-type compound according to item (6) above, whichcomprises one amino acid sequence selected from the group consisting ofamino acid sequences set forth in SEQ ID NOS: 3, 4, 5, 8, 9, 10, 11, 12,13, 16, 17, 18, 19, 22, 23, 25 and 26, or a pharmaceutically acceptablesalt thereof;

(8) The peptide-type compound according to item (1) or

(4) above, whose amino-terminal amino acids 1 to 4 are represented byformula:

A-B-C-D-

wherein the symbol A is either an amino acid or a non-amino acidcompound, or is missing, and the symbol B is either an amino acid or anon-amino acid compound, or is missing, provided that the length of theA+B molecular chain is a dipeptide length, and the symbol C or thesymbol D may be the same or different and represents (a) a modifiedamino acid, (b) an amino acid having a hydrophobic residue or (c) anamino acid having a basic side chain, or a pharmaceutically acceptablesalt thereof;

(9) A peptide-type compound, wherein in one amino acid sequence selectedfrom the group consisting of amino acid sequences set forth in SEQ IDNOS: 2, 3, 8, 9, 10, 11, 16, 17, 22, 25 and 26, an amino acid sequenceof amino-terminal amino acids 1 to 4 is s replaced by the structure ofthe peptide-type compound described in item (8) above, or apharmaceutically acceptable salt thereof.

(10) The peptide-type compound according to items (1) to (9) above,wherein the modified amino acid is an amino acid in which (a) asaturated or unsaturated alkyl chain containing one or more carbon atomswas introduced at the α carbon atom of the amino acid via or not via analkylene group containing one or more carbon atoms and via an ester,ether, thioester, thioether, disulfide, amide, carbamide orthiocarbamide linkage, or (b) a saturated or unsaturated alkyl chaincontaining one or more carbon atoms was introduced at the α carbon atomof the amino acid, or a pharmaceutically acceptable salt thereof;

(11) The peptide-type compound according to items (1) to (10) above,which comprises a modified amino acid modified by conversion of afunctional group in a side chain of said amino acid into an esterlinkage, or a pharmaceutically acceptable salt thereof;

(12) The peptide-type compound according to item (11) above, whichcomprises an amino acid having a fatty acid bound via an ester linkageto a side-chain hydroxyl group or mercapto group of said amino acid, ora pharmaceutically acceptable salt thereof;

(13) The peptide-type compound according to item (12) above, wherein thefatty acid contains 2 to 35 carbon atoms, or a pharmaceuticallyacceptable salt thereof;

(14) The peptide-type compound according to item (12) above, wherein thefatty acid is a fatty acid selected from the group consisting of fattyacids containing 2, 4, 6, 8, 10, 12, 14, 16 and 18 carbon atoms, or apharmaceutically acceptable salt thereof;

(15) The peptide-type compound according to item (12) above, wherein thefatty acid is octanoic acid, a monoene fatty acid thereof or a polyenefatty acid thereof, or a pharmaceutically acceptable salt thereof;

(16) The peptide-type compound according to item (12) above, wherein thefatty acid is decanoic acid, a monoene fatty acid thereof or a polyenefatty acid thereof, or a pharmaceutically acceptable salt thereof;

(17) The peptide-type compound according to items (1) to (16) above,wherein the amino-terminal amino group is modified by introduction of asaturated or unsaturated alkyl or acyl group containing one or morecarbon atoms, and/or a hydroxyl group of the carboxyl-terminal carboxylgroup is OZ or NR2R3 wherein Z is a pharmaceutically acceptable cationor a lower branched or linear alkyl group, and R2 and R3 are the same ordifferent and represent H or a lower branched or linear alkyl group;

(18) A peptide-type compound comprising a basic amino acid bound to thecarboxyl-terminal of a peptide-type compound described in items (1) to(16) above;

(19) A peptide-type compound comprising a basic group introduced into acarboxyl-terminal amide derivative of the peptide-type compounddescribed in items (1) to (16) or (18) above;

(20) A pharmaceutical composition comprising a peptide-type compounddescribed in items (1) to (19) above or a pharmaceutically acceptablesalt thereof as an active ingredient;

(21) A pharmaceutical composition for treatment of diseases attributableto a defect or decrease in growth hormone, which comprises apeptide-type compound described in items (1) to (19) above or apharmaceutically acceptable salt thereof as an active ingredient;

(22) A pharmaceutical composition for treatment of diseases notattributable to a defect or decrease in growth hormone, which comprisesan agent for treating diseases not attributable to a defect or decreasein growth hormone and a peptide-type compound described in items (1) to(19) above or a pharmaceutically acceptable salt thereof;

(23) A pharmaceutical composition according to items (20) to (22), whichis applied to animals other than human beings;

(24) A method for treatment of diseases attributable to a defect ordecrease in growth hormone, which comprises administering apharmaceutical composition comprising a peptide-type compound describedin items (1) to (19) above or a pharmaceutically acceptable salt thereofas an active ingredient;

(25) A method for treatment of diseases not attributable to a defect ordecrease in growth hormone, which comprises administering an agent fortreating diseases not attributable to a defect or decrease in growthhormone and a peptide-type compound described in items (1) to (19) aboveor a pharmaceutically acceptable salt thereof;

(26) A method according to item (24) or (25), which is applied toanimals other than human beings;

(27) A DNA coding an amino acid sequence of a peptide-type compounddescribed in items (1) to (19) above, which comprises a nucleotidesequence coding a peptide containing an amino acid sequence recognizingat least one modifiable amino acid in the amino acid sequence encoded bysaid DNA;

(28) The DNA according to item (27) above, wherein the nucleotidesequence is one nucleotide sequence selected from the group consistingof nucleotide sequences set forth in SEQ ID NOS: 6, 7, 14, 15, 20, 21and 24;

(29) The DNA according to item (27) above, wherein the nucleotidesequence is an amino acid-coding nucleotide sequence in one nucleotidesequence selected from the group consisting of nucleotide sequences setforth in SEQ ID NOS: 6, 7, 14, 15, 20, 21 and 24;

(30) A vector comprising a DNA described in items (27) to (29) above;

(31) Cells comprising the vector described in item (30) above;

(32) Cells comprising a DNA described in items (27) to (29) above,wherein a peptide-type compound having an amino acid sequence encoded bysaid DNA can be produced as a peptide-type compound having at least oneamino acid modified in said amino acid sequence;

(33) An antibody against a peptide-type compound described in items (1)to (19) above;

(34) A method for assaying a peptide-type compound described in items(1) to (19) above, which comprises using the antibody described in item(33) above to detect the peptide-type compound described in items (1) to(19) above;

(35) A kit for detecting a peptide-type compound described in items (1)to (19) above, which comprises using the antibody described in item (33)above to detect the peptide-type compound described in items (1) to (19)above;

(36) A method for producing a peptide-type compound described in items(1) to (19) above by genetic recombination technology, which comprisestransforming a vector containing a DNA described in items (27) to (29)above into host cells capable of modifying a side chain of at least oneamino acid in said peptide, then culturing the resulting transformedcells and recovering the desired peptide-type compound from the culture;

(37) A method for producing a peptide-type compound described in items(1) to (19) above by genetic recombination technology, which comprisestransforming a vector containing a DNA described in items (27) to (29)above into host cells, then culturing the resulting transformed cellsand recovering the desired compound from the culture, followed bychemically modifying an arbitrary amino acid thereof;

(38) A method for producing a peptide-type compound described in items(12) to (16) above by genetic recombination technology, which comprisesusing cells having the activity of binding a fatty acid via an esterlinkage to a side-chain hydroxyl group or a side-chain mercapto group ofan amino acid in the peptide-type compound;

(39) A method for producing a peptide-type compound described in items(12) to (16) above, which comprises using cells having the serineacylation activity of binding a fatty acid via an ester linkage to aside-chain hydroxyl group of serine in the amino acid sequence set forthin SEQ ID NO: 8;

(40) A pharmaceutical composition for gene therapy for treatment ofdiseases attributable to a defect or decrease in growth hormone, whichcomprises integrating a vector containing a DNA coding an amino acidsequence of a peptide-type compound described in items (1) to (19) aboveinto cells in a living body and expressing a peptide with at least onemodified amino acid, the peptide having the activity of increasing theintracellular calcium ion concentration;

(41) A method for treatment of diseases attributable to a defect ordecrease in growth hormone, which comprises integrating a vectorcontaining a DNA coding an amino acid sequence of a peptide-typecompound described in items (1) to (19) above into cells in a livingbody enabling a peptide having an amino acid sequence encoded by saidDNA to be produced as a peptide having an amino acid sequencerecognizing at least one modifiable amino acid in said amino acidsequence, whereby a peptide having the activity of inducing growthhormone is expressed;

(42) A pharmaceutical composition for gene therapy for treatment ofdiseases not attributable to a defect or decrease in growth hormone,which comprises integrating a vector containing a DNA coding an aminoacid sequence of a peptide-type compound described in items (1) to (19)above into cells in a living body and expressing a peptide with at leastone modified amino acid, the peptide having the activity of increasingthe intracellular calcium ion concentration;

(43) A method for treatment of diseases not attributable to a defect ordecrease in growth hormone, which comprises integrating a vectorcontaining a DNA coding an amino acid sequence of a peptide-typecompound described in items (1) to (19) above into cells in a livingbody enabling a peptide having an amino acid sequence encoded by saidDNA to be produced as a peptide having an amino acid sequencerecognizing at least one modifiable amino acid in said amino acidsequence, whereby a peptide having the activity of inducing growthhormone is expressed.

Specifically, the present invention also relates to:

(1) A peptide-type compound having the activity of increasing theintracellular calcium ion concentration, wherein at least one amino acidis replaced by a modified amino acid and/or a non-amino acid compound,or a pharmaceutically acceptable salt thereof;

(2) The peptide-type compound according to item (1) above, whichcomprises (a) an amino acid sequence set forth in SEQ ID NO: 2 or (b) anamino acid sequence having any one of amino acid sequences selected fromthe group consisting of

(1) amino acid sequence of amino acids 1 to 4,(2) amino acid sequence of amino acids 1 to 5,(3) amino acid sequence of amino acids 1 to 6,(4) amino acid sequence of amino acids 1 to 7,(5) amino acid sequence of amino acids 1 to 8,(6) amino acid sequence of amino acids 1 to 9, and(7) amino acid sequence of amino acids 1 to 10from the amino-terminal in the sequence (a) and at least one amino aciddeleted, replaced and/or added in a part outside said amino acidsequences, or a pharmaceutically acceptable salt thereof;

(3) The peptide-type compound according to item (2) above, whichcomprises one amino acid sequence selected from the group consisting ofamino acid sequences set forth in SEQ ID NOS: 3, 4, 5, 8, 9, 10, 11, 12,13, 16, 17, 18, 19, 22 and 23, or a pharmaceutically acceptable saltthereof;

(4) A peptide-type compound having the activity of increasing theintracellular calcium ion concentration and the activity of inducingsecretion of growth hormone, wherein (a) constitutional amino acids aremodified or not modified and (b) at least one amino acid is replaced ornot replaced by a non-amino acid compound, or a pharmaceuticallyacceptable salt thereof;

(5) The peptide-type compound according to item (4) above, whichcomprises (a) an amino acid sequence set forth in SEQ ID NO: 2 or (b) anamino acid sequence having any one of amino acid sequences selected fromthe group consisting of

(1) amino acid sequence of amino acids 1 to 4,(2) amino acid sequence of amino acids 1 to 5,(3) amino acid sequence of amino acids 1 to 6,(4) amino acid sequence of amino acids 1 to 7,(5) amino acid sequence of amino acids 1 to 8,(6) amino acid sequence of amino acids 1 to 9, and(7) amino acid sequence of amino acids 1 to 10from the amino-terminal in the sequence (a) and at least one amino aciddeleted, replaced and/or added in a part outside said amino acidsequences, or a pharmaceutically acceptable salt thereof;

(6) The peptide-type compound according to items (4) or (5) above, whichcomprises one amino acid sequence selected from the group consisting ofamino acid sequences set forth in SEQ ID NOS: 3, 4, 5, 8, 9, 10, 11, 12,13, 16, 17, 18, 19, 22 and 23, or a pharmaceutically acceptable saltthereof;

(7) The peptide-type compound according to items (1) to (6) above,wherein the modified amino acid is an amino acid at the 3rd positionfrom the amino-terminal thereof, or a pharmaceutically acceptable saltthereof;

(8) The peptide-type compound according to item (7) above, wherein theamino acid in the modified amino acid is serine or cysteine, or apharmaceutically acceptable salt thereof;

(9) The peptide-type compound according to items (1) to (6) above, whichcomprises a modified amino acid in which (a) a saturated or unsaturatedalkyl chain containing one or more carbon atoms was introduced at the αcarbon atom of the amino acid via or not via an alkylene groupcontaining one or more carbon atoms and via an ester, ether, thioester,thioether, amide or carbamide linkage, or (b) H or a saturated orunsaturated alkyl chain containing one or more carbon atoms wasintroduced at the α carbon atom of the amino acid, or a pharmaceuticallyacceptable salt thereof;

(10) The peptide-type compound according to items (1) to (6) above,which comprises a modified amino acid modified with an ester linkage, ora pharmaceutically acceptable salt thereof;

(11) The peptide-type compound according to item (10) above, whichcomprises an amino acid to which a fatty acid was bound, or apharmaceutically acceptable salt thereof;

(12) The peptide-type compound according to item (11) above, whichcomprises an amino acid to which a fatty acid containing 2 to 35 carbonatoms was bound, or a pharmaceutically acceptable salt thereof;

(13) The peptide-type compound according to item (12) above, whichcomprises an amino acid to which a fatty acid selected from the group ofconsisting fatty acids containing 2, 4, 6, 8, 10, 12, 14, 16 and 18carbon atoms was bound, or a pharmaceutically acceptable salt thereof;

(14) The peptide-type compound according to item (13) above, wherein thebound fatty acid is octanoic acid, a monoene fatty acid thereof or apolyene fatty acid thereof, or a pharmaceutically acceptable saltthereof;

(15) The peptide-type compound according to item (13) above, wherein thebound fatty acid is decanoic acid, a monoene fatty acid thereof or apolyene fatty acid thereo, or a pharmaceutically acceptable saltthereof;

(16) The peptide-type compound according to items (1) to (15) above,wherein the amino-terminal is modified with a saturated or unsaturatedalkyl or acyl group containing one or more carbon atoms, and/or ahydroxyl group of the carboxyl-terminal carboxyl group is OZ or NR2R3wherein Z is a pharmaceutically acceptable cation or a lower branched orlinear alkyl group, and R2 and R3 are the same or different andrepresent H or a lower branched or linear alkyl group;

(17) A pharmaceutical composition comprising a peptide-type compounddescribed in items (1) to (16) above or a pharmaceutically acceptablesalt thereof as an active ingredient;

(18) A pharmaceutical composition for treatment of diseases attributableto a defect or decrease in growth hormone, which comprises apeptide-type compound described in items (1) to (16) above or apharmaceutically acceptable salt thereof as an active ingredient;

(19) A pharmaceutical composition for treatment of diseases notattributable to a defect or decrease in growth hormone, which comprisesan agent for treating diseases not attributable to a defect or decreasein growth hormone and a peptide-type compound described in items (1) to(16) above or a pharmaceutically acceptable salt thereof;

(20) A pharmaceutical composition according to items (17) to (19), whichis applied to animals other than human beings;

(21) A method for treatment of diseases attributable to a defect ordecrease in growth hormone, which comprises administering apharmaceutical composition comprising a peptide-type compound describedin items (1) to (16) above or a pharmaceutically acceptable salt thereofas an active ingredient;

(22) A method for treatment of diseases not attributable to a defect ordecrease in growth hormone, which comprises administering an agent fortreating diseases not attributable to a defect or decrease in growthhormone and a peptide-type compound described in items (1) to (16) aboveor a pharmaceutically acceptable salt thereof;

(23) A method for treatment according to items (21) to (22), which isapplied to animals other than human beings;

(24) A DNA for a peptide-type compound described in items (1) to (16)above, which comprises a nucleotide sequence coding a peptide containingan amino acid sequence recognizing at least one modifiable amino acid inthe amino acid sequence encoded by said DNA;

(25) The DNA according to item (24) above, which comprises onenucleotide sequence selected from the group consisting of nucleotidesequences set forth in SEQ ID NOS: 6, 7, 14, 15, 20, 21 and 24;

(26) The DNA according to item (24) above, which comprises an aminoacid-coding nucleotide sequence in one nucleotide sequence selected fromthe group consisting of nucleotide sequences set forth in SEQ ID NOS: 6,7, 14, 15, 20, 21 and 24;

(27) A vector comprising a DNA described in items (24) to (26) above;

(28) Cells comprising the vector described in item (27) above;

(29) Cells comprising a vector containing a DNA described in items (24)to (26) above, wherein a peptide-type compound having an amino acidsequence encoded by said DNA can be produced as a peptide-type compoundhaving at least one amino acid modified in said amino acid sequence;

(30) An antibody against a peptide-type compound described in items (1)to (16) above;

(31) A method for assaying a peptide-type compound described in items(1) to (16) above, which comprises using the antibody described in item(30) above to detect the peptide-type compound described in items (1) to(16) above;

(32) A kit for detecting a peptide-type compound described in items (1)to (16) above, which comprises using the antibody described in item (30)above to detect said peptide-type compound;

(33) A method for producing a peptide-type compound described in items(1) to (16) above by genetic recombination technology, which comprisestransforming a vector containing a DNA described in items (24) to (26)above into host cells capable of modifying a side chain of at least oneamino acid in said peptide, then culturing the resulting transformedcells and recovering the desired peptide-type compound from the culture;

(34) A method for producing a peptide-type compound described in items(1) to (16) above by genetic recombination technology, which comprisestransforming a vector containing a DNA described in items (24) to (26)above into host cells, then culturing the resulting transformed cellsand recovering the desired peptide-type compound from the culture,followed by chemically modifying an arbitrary amino acid thereof;

(35) A method for producing the peptide-type compound described in items(11) to (15) above by genetic recombination technology, wherein thepeptide-type compound can be produced as a peptide having a fatty acidbound to a serine residue in the amino acid sequence set forth in SEQ IDNO: 8;

(36) A method for producing a peptide-type compound having the activityof increasing the intracellular calcium ion concentration and theactivity of inducing secretion of growth hormone, which comprisestransforming a vector containing a DNA coding a peptide-type compounddescribed in items (4) to (6) above into host cells, and culturing theresulting transformed cells and recovering the desired compound from theculture;

(37) A pharmaceutical composition for gene therapy for treatment ofdiseases attributable to a defect or decrease in growth hormone, whichcomprises integrating a vector containing a DNA coding a peptide-typecompound described in items (1) to (16) above into cells in a livingbody and expressing a peptide with at least one modified amino acid, thepeptide having the activity of increasing the intracellular calcium ionconcentration.

(38) A method for treatment of diseases attributable to a defect ordecrease in growth hormone, which comprises integrating a vectorcontaining a DNA coding a peptide-type compound described in items (1)to (16) above into cells in a living body enabling a peptide having anamino acid sequence encoded by said DNA to be produced as a peptidehaving an amino acid sequence recognizing at least one modifiable aminoacid in said amino acid sequence, whereby a peptide having the activityof inducing growth hormone is expressed;

(39) A pharmaceutical composition for gene therapy for treatment ofdiseases not attributable to a defect or decrease in growth hormone,which comprises integrating a vector containing a DNA coding apeptide-type compound described in items (1) to (16) above into cells ina living body and expressing a peptide with at least one modified aminoacid, the peptide having the activity of increasing the intracellularcalcium ion concentration;

(40) A method for treatment of diseases not attributable to a defect ordecrease in growth hormone, which comprises integrating a vectorcontaining a DNA coding a peptide-type compound described in items (1)to (16) above into cells in a living body enabling a peptide having anamino acid sequence encoded by said DNA to be produced as a peptidehaving an amino acid sequence recognizing at least one modifiable aminoacid in said amino acid sequence, whereby a peptide having the activityof inducing growth hormone is expressed.

Specifically, the present invention also relates to:

(1) A peptide-type compound having the activity of increasing theintracellular calcium ion concentration, wherein at least one amino acidis replaced by a modified amino acid and/or a non-amino acid compound,or a pharmaceutically acceptable salt thereof;

(2) The peptide-type compound according to item (1) above, whichcomprises an amino acid sequence set forth in SEQ ID NO: 1, or apharmaceutically acceptable salt thereof;

(3) The peptide-type compound according to item (1) above, whichcomprises an amino acid sequence set forth in SEQ ID NO: 2 or an aminoacid sequence wherein in SEQ ID NO: 2, at least one amino acid isdeleted, replaced and/or added in a part outside a sequence of aminoacids 1 to 7 from the amino-terminal thereof, or a pharmaceuticallyacceptable salt thereof;

(4) An analogue or derivative of the peptide described in item (1)above, which comprises an amino acid sequence set forth in SEQ ID NO: 3or an amino acid sequence wherein in SEQ ID NO: 3, at least one aminoacid is deleted, replaced and/or added in a part outside a sequence ofamino acids 1 to 7 from the amino-terminal thereof, or apharmaceutically acceptable salt thereof;

(5) A precursor peptide-type compound of the peptide compound describedin (3) above, which comprises an amino acid sequence set forth in SEQ IDNO: 4 or an amino acid sequence wherein in SEQ ID NO: 4, at least oneamino acid is deleted, replaced and/or added in a part outside asequence of amino acids 1 to 28 from the amino-terminal thereof;

(6) A precursor peptide-type compound of the peptide compound describedin item (4) above, which comprises an amino acid sequence set forth inSEQ ID NO: 5 or an amino acid sequence wherein in SEQ ID NO: 5, at leastone amino acid is deleted, replaced and/or added in a part outside asequence of amino acids 1 to 28 from the amino-terminal thereof;

(7) A peptide-type compound having the activity of increasing theintracellular calcium ion concentration and the activity of inducingsecretion of growth hormone, or a pharmaceutically acceptable saltthereof;

(8) The peptide-type compound according to item (7) above, which has theactivity of increasing the intracellular calcium ion concentration andthe activity of inducing secretion of growth hormone and has at leastone amino acid replaced by a non-amino acid compound, or apharmaceutically acceptable salt thereof;

(9) The peptide-type compound according to items (7) to (8) above, whichcomprises an amino acid sequence set forth in SEQ ID NO: 1, or aderivative thereof or a pharmaceutically acceptable salt thereof;

(10) The peptide compound described in items (7) to (8) above, whichcomprises an amino acid sequence set forth in SEQ ID NO: 2 or an aminoacid sequence wherein in SEQ ID NO: 2, at least one amino acid isdeleted, replaced and/or added in a part outside a sequence of aminoacids 1 to 7 from the amino-terminal thereof, or a derivative thereof ora pharmaceutically acceptable salt thereof;

(11) The peptide compound described in items (7) to (8) above, whichcomprises an amino acid sequence set forth in SEQ ID NO: 3 or an aminoacid sequence wherein in SEQ ID NO: 3, at least one amino acid isdeleted, replaced and/or added in a part outside a sequence of aminoacids 1 to 7 from the amino-terminal thereof, or a derivative thereof ora pharmaceutically acceptable salt thereof;

(12) A precursor peptide-type compound of the peptide-type compounddescribed in item (10) above, which comprises an amino acid sequence setforth in SEQ ID NO: 4 or an amino acid sequence wherein in SEQ ID NO: 4,at least one amino acid is deleted, replaced and/or added in a partoutside a sequence of amino acids 1 to 28 from the amino-terminalthereof;

(13) A precursor peptide-type compound of the peptide-type compounddescribed in item (11) above, which comprises an amino acid sequence setforth in SEQ ID NO: 5 or an amino acid sequence wherein in SEQ ID NO: 5,at least one amino acid is deleted, replaced and/or added in a partoutside a sequence of amino acids 1 to 28 from the amino-terminalthereof;

(14) The peptide-type compound according to items (1) to (6) above,wherein the modified amino acid is an amino acid at the 3rd positionfrom the amino-terminal thereof, or a pharmaceutically acceptable saltthereof;

(15) The peptide-type compound according to item (14), wherein the aminoacid in the modified amino acid is serine or cysteine, or apharmaceutically acceptable salt thereof;

(16) The peptide-type compound according to items (1) to (6) above,wherein the modification in the modified amino acid indicates themodification at the α carbon of said amino acid by (a) a saturated orunsaturated alkyl chain containing one or more carbon atoms which bindsin a mode of linkage selected from the group consisting of ester, ether,thioester, thioether, amide or carbamide via or not via an alkyl chain,containing one or more carbon atoms, or (b) H or a saturated orunsaturated alkyl chain containing one or more carbon atoms, or apharmaceutically acceptable salt thereof;

(17) The peptide-type compound according to item (1) above, whichcomprises a modified amino acid modified with an ester linkage, or apharmaceutically acceptable salt thereof;

(18) The peptide-type compound according to item (17) above, whichcomprises an amino acid to which a fatty acid is bound, or apharmaceutically acceptable salt thereof;

(19) The peptide-type compound according to item (18) above, whichcomprises an amino acid to which a fatty acid containing 2 to 35 carbonatoms is bound, or a pharmaceutically acceptable salt thereof;

(20) The peptide-type compound according to item (18) above, wherein thebound fatty acid is caprylic acid, a monoene fatty acid thereof or apolyene fatty acid thereof, or a pharmaceutically acceptable saltthereof;

(21) The peptide-type compound according to item (18) above, wherein thebound fatty acid is capric acid, a monoene fatty acid thereof or apolyene fatty acid thereof, or a pharmaceutically acceptable saltthereof;

(22) The peptide-type compound according to item (18) above, wherein thebound fatty acid is lauric acid, a monoene fatty acid thereof or apolyene fatty acid thereof, or a pharmaceutically acceptable saltthereof;

(23) The peptide-type compound according to items (1) to (22) above,wherein the amino-terminal is modified with a saturated or unsaturatedalkyl or acyl group containing one or more carbon atoms, and/or thecarboxyl-terminal is OZ or NR2R3 wherein Z is a pharmaceuticallyacceptable cation or a lower branched or linear alkyl group, and R2 andR3 are the same or different and represent H or a lower branched orlinear alkyl group;

(24) A pharmaceutical composition for treatment of diseases attributableto a defect or decrease in growth hormone, which comprises apeptide-type compound described in items (1) to (6) above or apharmaceutically acceptable salt thereof as an active ingredient;

(25) A pharmaceutical composition for treatment of diseases notattributable to a defect or decrease in growth hormone, which comprisesan agent for treating diseases not attributable to a defect or decreasein growth hormone and a peptide-type compound described in items (1) to(6) above or a pharmaceutically acceptable salt thereof;

(26) A pharmaceutical composition according to items (24) to (25), whichis applied to animals other than human beings;

(27) A method for treatment of diseases attributable to a defect ordecrease in growth hormone, which comprises administering apharmaceutical composition comprising a peptide-type compound describedin items (1) to (6) above or a pharmaceutically acceptable salt thereofas an active ingredient;

(28) A method for treatment of diseases not attributable to a defect ordecrease in growth hormone, which comprises administering an agent fortreating diseases not attributable to a defect or decrease in growthhormone and a peptide-type compound described in items (1) to (6) aboveor a pharmaceutically acceptable salt thereof;

(29) The treatment method according to items (27) to (28), which isapplied to animals other than human beings;

(30) A DNA for a peptide-type compound described in items (1) to (6),which comprises a DNA sequence coding a peptide having a amino acidsequence recognizing at least one modifiable amino acid in said aminoacid sequence;

(31) The cDNA according to item (30) above, which comprises a DNAsequence set forth in SEQ ID NO: 6 (including NCR);

(32) The cDNA according to item (30) above, which comprises a DNAsequence of bases 31 to 381 in a DNA sequence set forth in SEQ ID NO: 6(not including NCR);

(33) The cDNA according to item (30) above, which comprises a DNAsequence set forth in SEQ ID NO: 7 (including NCR);

(34) The cDNA according to item (30) above, which comprises a DNAsequence of bases 34 to 385 in a DNA sequence set forth in SEQ ID NO: 7(not including NCR);

(35) A vector comprising a DNA described in items (30) to (34) above;

(36) Cells comprising the vector described in item (35) above;

(37) Cells comprising a vector containing a DNA described in items (30)to (34) above, wherein a peptide-type compound having an amino acidsequence encoded by said DNA can be produced as a peptide-type compoundhaving an amino acid sequence recognizing at least one modifiable aminoacid in said amino acid sequence;

(38) An antibody against a peptide-type compound described in items (1)to (23) above;

(39) A method for assaying a peptide-type compound described in items(1) to (23) above, which comprises using the antibody described in item(38) above to detect the peptide-type compound;

(40) A kit for detecting a peptide-type compound described in items (1)to (23) above, which comprises using the antibody described in item (38)above to detect the peptide-type compound;

(41) A method for producing a peptide-type compound described in items(1) to (6) above by genetic recombination technology, which comprisestransforming a vector containing a DNA described in item (30) above intohost cells capable of modifying a side chain of at least one amino acidin said peptide, then culturing the resulting transformed cells andrecovering the desired peptide-type compound from the culture;

(42) A method for producing a peptide-type compound described in items(1) to (6) above by genetic recombination technology, which comprisestransforming a vector containing a DNA described in item (30) above intohost cells, then culturing the resulting transformed cells andrecovering the desired peptide-type compound from the culture, followedby chemically modifying an arbitrary amino acid thereof;

(43) A method for producing a peptide-type compound described in items(18) to (22) above by genetic recombination technology, which comprisesusing cells enabling the peptide-type compound to be produced as apeptide having a fatty acid bound to a serine residue in the amino acidsequence set forth in SEQ ID NO: 1;

(44) A method for producing a peptide-type compound having the activityof increasing the intracellular calcium ion concentration and theactivity ity of secreting growth hormone, which comprises transforming avector containing a DNA coding a peptide-type compound described initems (7) to (13) above into host cells, culturing the resultingtransformed cells and recovering the desired compound from the culture;

(45) A pharmaceutical composition for gene therapy for treatment ofdiseases attributable to a defect or decrease in growth hormone, whichcomprises integrating a vector containing a DNA coding an amino acidsequence of a peptide-type compound described in items (1) to (6) aboveinto cells in a living body and expressing a peptide with at least onemodified amino acid, the peptide having the activity of increasing theintracellular calcium ion concentration;

(46) A method for treatment of diseases attributable to a defect ordecrease in growth hormone, which comprises integrating a vectorcontaining a DNA coding an amino acid sequence of a peptide-typecompound described in items (1) to (6) above into cells in a living bodyenabling a peptide having an amino acid sequence encoded by said DNA tobe produced as a peptide having an amino acid sequence recognizing atleast one modifiable amino acid in said amino acid sequence, whereby apeptide having the activity of inducing growth hormone is expressed;

(47) A pharmaceutical composition for gene therapy for treatment ofdiseases not attributable to a defect or decrease in growth hormone,which comprises integrating a vector containing a DNA coding an aminoacid sequence of a peptide-type compound described in items (1) to (6)above into cells in a living body and expressing a peptide with at leastone modified amino acid having the activity of increasing theintracellular calcium ion concentration;

(48) A method for treatment of diseases not attributable to a defect ordecrease in growth hormone, which comprises integrating a vectorcontaining a DNA coding an amino acid sequence of a peptide-typecompound described in items (1) to (6) above into cells in a living bodyenabling a peptide having an amino acid sequence encoded by said DNA tobe produced as a peptide having an amino acid sequence recognizing atleast one modifiable amino acid in said amino acid sequence, whereby apeptide having the activity of inducing growth hormone is expressed.

In the present invention, the amino acid encompasses every amino acidsuch as L-amino acid, D-amino acid, α-amino acid, β-amino acid, γ-aminoacid, natural amino acid and synthetic amino acid or the like.

In the present invention, the modified amino acid refers to an aminoacid wherein an arbitrary group thereof is chemically modified. Inparticular, a modified amino acid chemically modified at the α-carbonatom in an α-amino acid is preferable. That is, when the α-amino acid isrepresented by formula (1):

R′ and R″ in the chemically modified amino acid may be H or an arbitrarygroup; in short, the modified amino acid may be any chemically modifiednatural amino acid. Either R′ or R″ may also be H.

An amino acid wherein as the substituent group represented by R′ and R″,a substituent group present in the natural amino acid is replaced by asubstituent group not present in the natural amino acid or in itscorresponding D-amino acid is referred to as the modified amino acid.

When the naturally occurring amino acid contains e.g. —OH, —SH, —NH or—NH₂ as a substituent group in a side chain thereof, a group formed byacylating such a substituent group is mentioned as a preferable exampleof the subtituent group mentioned above.

The acyl group therefor includes e.g. groups formed by removing ahydroxyl group from an organic carboxylic acid, organic sulfonic acidand organic phosphoric acid.

The organic carboxylic acid includes e.g. fatty acids, and the number ofcarbon atoms thereof is preferably 2 to 35, more preferably 6 to 18 andmost preferably 8 to 16. Such fatty acids include e.g. octanoic acid(preferably caprylic acid), decanoic acid (preferably capric acid), anddodecanoic acid (preferably lauric acid), as well as monoene or polyenefatty acids thereof.

In the organic sulfonic acid or organic phosphoric acid, the number ofcarbon atoms thereof is preferably 2 to 35.

Further, the modified amino acid may be an amino acid wherein the grouprepresented by R′ and/or R″ is replaced, for example, by:

—(CH₂)_(n)-P-Q

(wherein n is an integer of 0 to 10, P is —CO—O—, —O—CO—, —O—, —CO—S—,—CS—S—, —S—CO—, —S—, —CO—NH—, —NH—CO— or —CO—NH—CO—, Q is H or C₁₋₃₅preferably C₁₋₂₀, alkyl.) Further, P may be —CO—.

In addition, P may be —S—S— or —NH—CS—. In every —NH— described above, Hmay be replaced by a C₁₋₃₅ saturated or unsaturated alkyl group, a C₆₋₂₀aryl group, or a C₇₋₁₃ aralkyl group.

In the case where the α-amino acid is represented by the formula (1)above, the modified amino acid wherein R′ or R″ is replaced by the abovegroup —(CH₂)_(n)-P-Q is a preferable embodiment. In particular, saidmodified amino acid is preferably modified serine wherein a substituentgroup represented by the above formula —(CH₂)_(n)-P-Q is bound to theα-carbon of serine, as shown in the formula:

wherein n, P and Q have the same meanings as defined above.

The mode of linkage selected from the group consisting of ester, ether,thioester, thioether, amide and carbamide via or not via an alkyl groupcontaining one or more carbon atoms is described in more detail.

For example, if the amino acid is serine, threonine, tyrosine oroxyproline, the amino acid has a hydroxyl group in the side chain. Ifthe amino acid is cysteine, the amino acid has a mercapto group in theside chain. If the amino acid is lysine, arginine, histidine,tryptophan, proline or oxyproline, it has an amino group or imino groupin the side chain.

The hydroxyl group, mercapto group, amino group and imino groupdescribed above may have been chemically modified. That is, the hydroxylgroup or mercapto group may be etherized, esterified, thioetherified orthioesterified. The imino group may have been iminoetherified,iminothioetherified or alkylated. The amino group may have beenamidated, thioamidated or carbamidated.

Further, the mercapto group may have been disulfidated, the imino groupmay have been amidated or thioamidated, and the amino group may havebeen alkylated or thiocarbamidated.

The thus chemically modified hydroxyl group or mercapto group can berepresented for example by:

The amidated or thioamidated amino group or imino group can berepresented by:

The etherified hydroxyl group or mercapto group can be represented by:

—O—Z₃, or

—S—Z₃

The iminoetherified or iminothioetherified imino group can berepresented by:

The alkylated amino group can be represented by:

The alkylated imino group can be represented by:

The carbamidated or thiocarbamidated imino group can be represented by:

The disulfidated mercapto group can be represented by: —S—S—Z₈

In the formulae above, Z₁, Z₂, Z₃, Z₄, Z₅, Z₆, Z₇ and Z₈ may be anysubstituent groups for chemical modification insofar as they are notagainst the sprit of the present invention, but because substituentgroups used conventionally in a pharmaceutical field or for chemicalmodification of peptides are well known in patent literatures orscientific literatures, such known substituent groups for modificationcan be used and according to such known methods, chemical modificationcan be performed in the present invention.

In the above formulae, Z₁ may be a hydrogen atom or a linear-chain,branched or cyclic alkyl group, and such alkyl group may be saturated orunsaturated. The number of carbon atoms thereof is usually C₁₋₅₀,preferably C₆₋₂₀.

Z₂, Z₃, Z₄, Z₅, Z₆, Z₇ or Z₈ may be a hydrogen atom or a straight-chain,branched or cyclic alkyl group, and such alkyl group may be saturated orunsaturated. The number of carbon atoms thereof is usually C₁₋₁₀,preferably C₁₋₆.

The alkyl groups represented by Z₁, Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, or Z₈ may besubstituted with substituent groups such as hydroxyl group, amino group,halogen, nitro and C₁₋₃ alkoxy group, which are used conventionally forchemical modification of peptides.

In the above, if Z₁—CO— is a residue of the fatty acid Z₁—COOH, this isone example of the amino acid to which the fatty acid was bound. Thefatty acid in this case includes e.g. saturated fatty acid such ascaprylic acid, capric acid, lauric acid, butyric acid, caproic acid,undecylic acid, palmitic acid, decanoic acid, nonadecanoic acid, behenicacid, montanic acid and lacceric acid and unsaturated fatty acid such asacrylic acid, oleic acid, linolic acid, linolenic acid and aatearolicacid. The unsaturated fatty acid may be a monoene or a polyene.

Further, the modified amino acid may also be an α-amino acid formed byreplacing a group (excluding a carboxyl group and an amino groupconstituting a peptide linkage) binding to the α-carbon atom of theα-amino acid by a hydrogen atom or a saturated or unsaturated alkylgroup.

In the present invention, the modified amino acid may also be an aminoacid formed by introducing a C₁₋₆ saturated or unsaturated alkyl grouponto the amino group of the amino acid.

The non-natural amino acid in the present invention is the one havingamino group and carboxyl group in both terminals of the molecule, andincludes e.g. NH₂—(CH₂)₃CH(CH₂OH)—COOH, NH₂—(CH₂)₄—COOH,NH₂—C(CH₃)₂—(CH₂)₃—COOH, and NH₂—CH(CH₃)—(CH₂)₂—CH(CH₃)—COOH. The lengthof their molecular chain corresponds to the length of a dipeptide, butthe non-natural amino acid in the present invention also includes thosehaving the length of a peptide.

Further, the non-amino acid compound in the present invention includese.g. NH₂—CH(CH₂OH)—CH₃, CH₃—CH(R)—COOH, CH₃—CH(R)—CH₃ wherein the lengthof their molecule corresponds to the length of a peptide, orNH₂—(CH₂)₃CH(CH₂OH)—CH₃ and NH₂—(CH₂)₃CH(R)—CH₃ wherein the length oftheir molecule corresponds to the length of a dipeptide.

Here, R represents a substituent group on a side chain of the naturalamino acid or on the α-carbon in the aforementioned modified amino acid.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows purification of ghrelin from rat stomach extract, and thechange in fluorescence intensity by an increase in the intracellularcalcium ion concentration in CHO-GHSR62 cells is shown by the black bar.FIG. 1 a shows a profile in Sephadex G-50 (fine) gel filtration of anSP-III fraction prepared from 40 g rat stomach, to indicate that themolecular weight of the active fractions is about 3,000 Dalton. FIG. 1 bis a graph shows a profile in secondary CM ion-exchange HPLC, and theactive fractions eluted at retention times of 55 to 56 minutes werefurther purified by reversed-phase HPLC.

FIG. 2 shows that the modification of ghrelin with n-octanoyl wasidentified. FIG. 2 a shows the result of analysis of 2 μg each ofnatural ghrelin (upper) and synthetic ghrelin and synthetic de-acylatedghrelin (lower) by reversed-phase HPLC. FIG. 2 b is a graph showingchanges in the intracellular calcium ion concentration in CHO-GHSR62cells by natural ghrelin (solid line), synthetic ghrelin (small brokenline) and synthetic de-acylated ghrelin (large solid line).

FIG. 3 is a graph showing specific interaction of ghrelin withCHO-GHSR62 cells, and ghrelin was added at the arrowed point. FIG. 3 ais a graph showing changes in the intracellular calcium ionconcentration in CHO-GHSR62 cells by ghrelin, GHRP-6 and GRF (GHRH),respectively. Fig. b is a graph showing changes in the intracellularcalcium ion concentration in CHO-GHSR62 cells by ghrelin in the presence(◯) or absence () of [D-Lys-3]-GRP-6, and a change in the intracellularcalcium ion concentration by GRF (GHRH) (black triangle) is also shown.

FIG. 4 shows the amino acid sequences of rat- and human-derived ghrelinprecursors, as well as the analysis result of expression of theseprecursors in various tissues. FIG. 4 a shows comparison between theamino acid sequences of rat- and human-derived ghrelin precursors, wherethe same amino acid is shaded, a signal peptide is indicated by thebroken line, a cleavage site of the signal peptide is indicated by theshaded triangle, a cleavage site at the side of carboxyl-terminal isindicated by the triangle, a matured ghrelin moiety is boxed, and amodification with n-octanoic acid is indicated by *. FIG. 4 b shows theanalysis result of expression of ghrelin in a wide variety of rattissues by Northern blotting.

FIG. 5 is a graph showing the effect of ghrelin in vitro and in vivo onsecretion of pituitary hormones. FIG. 5 a is a graph showing a change influorescence intensity by a change in the intracellular calcium ionconcentration in rat pituitary cultured cells at an initial stage, wherethe change upon addition of ghrelin is indicated by the solid line andthe change upon addition of de-acylated ghrelin by the broken line. FIG.5 b is a graph showing the secretion of pituitary hormones, where theblack bar and white bar show the concentrations of pituitary hormonelevels in the presence and absence of ghrelin, respectively. FIG. 5 c isa graph showing a time course of pituitary hormone concentration inplasma after ghrelin was injected intravenously to male rats. In FIGS. 5b and 5 c, GH is growth hormone, ACTH is adrenocorticotropin, FSH isfollicle-stimulating hormone, LH is luteinizing hormone, PRL isprolactin, and TSH is thyroid-stimulating hormone.

FIG. 6 shows promotion of appetite upon administration of ghrelin intoventricle, where the amount of feed (mean±standard error) for 2 hoursafter administration of ghrelin is shown. FIG. 6 a shows that the errorrange for the effect of ghrelin is less than 0.0001.

FIG. 7 shows the effect of a drug administered to rat under urethaneanesthesia on secretion of gastric acid, where A and B show the resultsof administration of rat ghrelin (rGhrelin) and histamine, respectively.Each symbol indicates an average value from 4 rats, and the standarderror is shown by an error bar. As the control, physiological saline wasadministered. At the arrowed point, the drug was administered.

FIG. 8 is a graph showing the action of rat ghrelin on stomach motilityin rat under urethane anesthesia. FIG. 8A shows typical waves of stomachmotility upon administration of physiological saline and rat ghrelin(rGhrelin), and Fig. B is a graph showing an average value from 4 ratsalong with the standard error. At the arrowed point, the drug wasadministered.

FIG. 9 is a graph showing a standard curve in radioimmunoassays andcross-reactivity with antibody. FIG. 9 a is a graph showing the bindinginhibition, by various ghrelins, of ¹²⁵I-labeled rat ghrelin to anantibody against a amino-terminal ghrelin fragment, and FIG. 9 b is agraph showing the binding inhibition, by various ghrelins, of¹²⁵I-labeled rat ghrelin to an antibody against a carboxyl-terminalghrelin fragment. The amount of various ghrelins/reaction tube is shownon the abscissa, while the ratio (%) of the amount (B) of rat ghrelinbound in the presence of various ghrelins to the amount thereof (B₀) inthe absence of various ghrelins is shown on the ordinate. Symbols in thegraphs are as follows: Rat ghrelin (◯); human ghrelin (); ratghrelin-27 (□); [Ser3 (decanoyl)]-rat ghrelin (⋄); [Ser3 (hexanoyl)]-ratghrelin (Δ); and de-fatty acid rat ghrelin (▾).

BEST MODE FOR CARRYING OUT THE INVENTION

For a peptide serving as an endogenous ligand for GHS receptor (GHS-R),the distribution of the endogenous ligand in organs or tissues can beknown by adding an extract from various organs or tissues to cellsexpressing GHS-R and measuring the intracellular calcium ionconcentration.

The cells expressing GHS-R include strains derived from the hypothalamusand pituitary gland known to express GHS-R constantly and their tissues,but the cells are preferably those transformed cells having GHS-R geneintroduced into suitable cells such as CHO cells, and expressing thegene.

The strong Ca-releasing activity of the endogenous GHS peptide of thepresent invention was found not in the hypothalamus and pituitary glandexpressing the peptide, but in an extract from stomach as an organ inthe digestive organ system. It is therefore necessary to examine notonly tissues and organs expressing said receptor but also a wide varietyof other tissues and organs in order to find the desired endogenousligand for the orphan receptor.

The intracellular calcium ion concentration can be measured by anymethod known in the art, preferably by means of FLIPR (FluorometricImaging Plate Reader, Molecular Devices Co., Ltd.) utilizing the changein the fluorescent intensity of Fluo-4 AM (Molecular Probe Co., Ltd.)caused by a change in the concentration of calcium ions.

To obtain the desired endogenous GHS peptide from tissues and organsconfirmed to exhibit the Ca-releasing activity, any purification methodknown in the art can be used.

As the method of purifying the peptide, it is effective to use acombination of gel filtration, ion exchange and reversed-phasechromatographic techniques after a wide variety of fractionation methodsor use them separately, but it is possible to use not only suchchromatographic techniques but also any means effective for purificationof the peptide.

For isolation and purification of the peptide from the tissues andorgans, inactivation of proteases in the tissues and organs by heattreatment thereof in boiling water is desired to prevent degradation ofthe desired peptide by the action of the proteases. Heat treatment andremoval of the tissues and organs under cooling on ice are alsoeffective for extraction and purification of the desired peptide.

To confirm that the purified peptide having the Ca-releasing activityhas a GH secretion-inducing activity in vitro and in vivo, a knownmethod can be utilized.

For example, GH secreted into a medium of pituitary grand cell cultureconfirmed to secrete GH and express GHS-R can be measured in vitro inradioimmunoassays by adding anti-GH antibody to the cells. By using anantibody against other hormone in place of the anti-GH antibody inradioimmunoassays, the amount of said hormone secreted can also bemeasured.

Further, the GH secretion-inducing activity in vivo can be confirmed byinjecting the peptide having the Ca-releasing activity into a peripheralvein of an animal and then measuring the concentration of GH in serum.

For analyzing the structure of the purified peptide, a known method canbe used.

For determining the amino acid sequence of the peptide, there is amethod wherein amino acid residues are released sequentially from thecarboxyl-terminal by Edman degradation followed by identification of thereleased amino acids by high performance liquid chromatography (HPLC),as well as an automated version thereof by an amino acid sequencer.

There is also a method for determining the amino acid sequence thereofby measuring the molecular weights of ionized fragments thereof byGC-MASS.

For the peptide containing modified amino acids in one aspect of thepresent invention, the modified amino acid is identified as “unknownamino acid” upon determination of the amino acid sequence.

In this case, the modified peptide is decomposed into amino acid unitsfrom which the modified amino acid is separated and purified, and thestructure of the modified amino acid is determined in a usual manner fordetermining the structure of the compound, whereby the entire structureof the peptide can be known. Alternatively, there is a method whereinthe peptide is obtained from a cDNA coding the modified peptide, then apeptide having the amino acid sequence of the resulting peptide ischemically synthesized, and the molecular weight and physical propertiesof the synthetic unmodified peptide are compared with those of themodified peptide in order to estimate the structure of the modifiedgroup.

A partial amino acid sequence (core sequence) which in the peptide thusstructurally determined, is essential for the Ca-releasing activity isrevealed by measuring the Ca-releasing activity of each peptide fragmentformed by cleaving said peptide with a protease.

The used protease shall be a protease highly specific to the amino acidsequence of the peptide to be cleaved, but a low specific protease canalso be used under conditions for partial digestion to prepare variouspeptide fragments from said peptide.

By measuring the Ca-releasing activity of each peptide fragment thusprepared, a core sequence essential for the Ca-releasing activity can beknown.

In the endogenous GH secretion-inducing peptide, serine 3 from theamino-terminal has been acylated with a fatty acid, and it is alsopossible to chemically be synthesized a peptide fragment having apart ofthe amino acid sequence of the endogenous GH secretion-inducing peptide,as well as a fatty acid-modified peptide comprising a fatty acid boundvia an ester linkage to the serine side chain in said peptide fragment.

Using said peptide fragment, the endogenous GH secretion-inducingpeptide can be analyzed in detail. Simultaneously, the type of fattyacid necessary for the Ca-releasing activity can be determined bycomparing the peptide fragments modified with various fatty acids.

For example, in the endogenous GH secretion-inducing peptide derivedfrom some species of Xenopus Laevis, an amino acid residue at the 3rdposition from the amino-terminal is not serine but threonine, and suchthreonine has been acylated with a fatty acid, and this peptide-typecompound can also be synthesized and said compound can be analyzed indetail.

By comparing the amino acid sequences of those peptides having a GHsecretion-inducing activity in vertebrates, a region preserved widely invertebrates can be found, and from the amino acid sequence of saidregion, a core sequence essential for the GH secretion-inducing activitycan be found.

A DNA having a nucleotide sequence deduced from the amino acid sequenceof the endogenous GH secretion-inducing peptide is chemicallysynthesized, and this DNA is used as a probe for screening a cDNAlibrary prepared from mRNA in cells expressing said peptide, whereby acDNA coding said peptide can be obtained.

However, a codon corresponding to an amino acid is degenerated thusincreasing the number of nucleotide sequences deduced from the aminoacid sequence of the peptide, so that the screening by using a certainsynthetic DNA consisting of various types of such nucleotide sequencesas a probe can be difficult.

In this case, if a sequence in accordance with the amino acid sequenceof said peptide is present in amino acid sequences deduced from thenucleotide sequence of an expressed sequence tag (EST) disclosed in asequence data base, a DNA consisting of a part of the nucleotidesequence of the EST can be synthesized and used to screen the above cDNAlibrary.

Further, a genomic DNA can be obtained in a usual manner from the cDNA.

From the nucleotide sequence of the cDNA thus obtained, the amino acidsequence of a precursor polypeptide of the endogenous GHsecretion-inducing peptide is revealed.

By analyzing said amino acid sequence, a signal peptide, the endogenousGH secretion-inducing peptide, other peptide moieties and cleavage sitesof these peptides are revealed, thus revealing the mechanism offormation of the endogenous GH secretion-inducing peptide.

Other aspects of the present invention, that is, a partial amino acidsequence of the endogenous GH secretion-inducing peptide, the amino acidsequence of a precursor polypeptide of said peptide, and the nucleotidesequence of a DNA coding said polypeptide are disclosed in InternationalAppln. Disclosure WO 98/42840, but the peptide disclosed therein is apeptide consisting of 14 amino acids having a motilin-like activity, andthere is no description therein of the activity of increasing Caconcentration and the activity of inducing GH secretion disclosed in thepresent invention.

The peptide-type compound of the present invention refers to a peptidehaving the activity of increasing the intracellular calcium ionconcentration, which is represented by formula (2) below wherein atleast one amino acid is replaced by a modified amino acid; a peptideanalogue thereof wherein at least one amino acid is replaced by anon-amino acid; and a peptide derivative thereof wherein amino-terminaland/or carboxyl-terminal is modified.

In the present invention, the peptide, peptide analogue and peptidederivative described above are referred to collectively as thepeptide-type compound.

In the peptide-type compound, a plurality of amino acids may be replacedby modified amino acids and/or non-amino acids. In the amino acidsequence set forth in SEQ ID No:2, it is preferable in the presentinvention that usually one or more amino acids of amino acids 1 to 10from the amino-terminal, preferably amino acids 1 to 4 or amino acids 1to 5 from the amino-terminal are replaced by modified amino acids and/ornon-amino acids. It is particularly preferable that amino acids 1 to 5are replaced by modified amino acids and/or non-amino acids.

In the amino acid sequence set forth in SEQ ID NO: 2, one or more aminoacids outside of amino acids 1 to 4, preferably amino acids 1 to 6 andmore preferably amino acids 1 to 10 from the amino-terminal may be addedor deleted.

The peptide-type compound of the present invention is preferably apeptide compound which has the activity of increasing the intracellularcalcium ion concentration and induces secretion of growth hormone invivo, wherein at least one amino acid is replaced by a modified aminoacid and/or a non-amino acid compound.

That is, the peptide-type compound of the present invention is apeptide-type compound having the activity of increasing theintracellular calcium ion concentration and/or the action of inducingsecretion of growth hormone in vivo, wherein an amino acid in thepeptide chain is replaced by a modified amino acid and/or a non-aminoacid compound.

Examples of the compound include those compounds wherein in the peptideshown in SEQ ID NO: 1, 2 or 3, a hydroxyl group of the amino acid Ser 3is acylated, those compounds wherein in the peptide shown in SEQ ID NO:4 or 5, a hydroxyl group of the amino acid Ser 25 is acylated, orpharmaceutically acceptable salts thereof.

Other examples include those compounds wherein in the peptide shown inSEQ ID NO: 10, 11, 16 or 17, a hydroxyl group of the amino acid Ser 3 isacylated, or pharmaceutically acceptable salts thereof.

Still other examples include those compounds wherein in the peptideshown in SEQ ID NO: 22, 25, 26 or 27, a hydroxyl group of the amino acidSer 3 is acylated, or pharmaceutically acceptable salts thereof.

Still other examples include those compounds wherein in the peptideshown in SEQ ID NO: 29, 30 or 31, a hydroxyl group of the amino acid Ser3 is acylated, or pharmaceutically acceptable salts thereof.

Still other examples include those compounds wherein in the peptideshown in SEQ ID NO: 28, a hydroxyl group of the amino acid Thr 3 isacylated, or pharmaceutically acceptable salts thereof.

The acyl group introduced into a hydroxyl group by acylation in thepresent invention is a group formed by removing a hydroxyl group frome.g. an organic carboxylic acid, an organic sulfonic acid or an organicphosphoric acid.

The organic carboxylic acid includes e.g. fatty acids, and the number ofcarbon atoms thereof is preferably about 2 to 35, more preferably about6 to 18, and most preferably about 8 to 16. Such fatty acids includee.g. octanoic acid (preferably caprylic acid), decanoic acid (preferablycapric acid), and dodecanoic acid (preferably laurylic acid [sic: lauricacid]), as well as their monoene or polyene fatty acids thereof.

In the organic sulfonic acid or organic phosphoric acid, the number ofcarbon atoms thereof is preferably about 2 to 35.

Any peptide-type compounds or pharmaceutically acceptable salts thereof,including the amino acid sequence set forth in SEQ ID NO: 1 wherein ahydroxyl group of Ser 3 is acylated, are also preferable embodiments ofthe present invention.

That is, in the second aspect of the present invention, any peptide-typecompounds or pharmaceutically acceptable salts thereof, including fattyacid-modified peptides wherein a hydroxyl group of Ser 3 is acylated inthe amino acid sequence set forth in SEQ ID NO: 8, preferably the aminoacid sequence set forth in SEQ ID NO: 1 and more preferably the aminoacid sequence set forth in SEQ ID NO: 9, are also preferable embodimentsof the present invention.

Further, any peptide compounds or pharmaceutically acceptable saltsthereof, including fatty acid-modified peptides wherein a hydroxyl groupof Thr 3 is acylated in the amino acid sequence set forth in SEQ ID NO:8, preferably the amino acid sequence set forth in SEQ ID NO: 1 and morepreferably an amino acid sequence wherein in the amino acid sequence setforth in SEQ ID NO: 9, the amino acid residue serine at the 3rd positionfrom the amino-terminal is converted into threonine, are also preferableembodiments of the present invention.

Further, a preferable embodiment of the present invention is a compoundor pharmaceutically acceptable salts represented by formula (2):

X-AA1-AA2-AA3-Y  (2)

wherein X is a moiety corresponding to a hydrogen atom in an amino groupof the amino-terminal amino acid and represents H or a saturated orunsaturated alkyl or acyl group containing one or more carbon atoms; Yis a moiety corresponding to a hydroxyl group in an α-carboxyl group ofthe carboxyl-terminal amino acid and represents OH, OZ or NR6R7whereupon Z is a pharmaceutically acceptable cation or a lower branchedor linear alkyl group; and R6 and R7 may be the same or different andrepresent H or a lower branched or linear alkyl group.

Here, AA1 represents:

wherein n is 1 or 2, R₁ and R₁′ may be the same or different andrepresent hydrogen or a substituent group, provided that when n is 2,the two substituent groups R₁ may be the same or different; this alsoapplies to R₁′.

Examples of the substituent group include (1) a saturated or unsaturatedalkyl chain containing one or more carbon atoms which binds in a mode oflinkage selected from the group consisting of ester, ether, thioester,thioether, amide and carbamide via or not via an alkyl chain containingone or more carbon atoms, (2) H or a saturated or unsaturated alkylchain containing one or more carbon atoms, and (3) a side chain ofnatural amino acid.

Further, the substituent group may be a saturated or unsaturated alkylchain containing one or more carbon atoms, which is bound via adisulfide or thiocarbamide linkage via or not via an alkyl chaincontaining one or more carbon atoms.

AA2 represents:

wherein R₁ and R₁′ have the same meanings as defined above, and R₂represents H or a saturated or unsaturated alkyl group containing 1 to 6carbon atoms, or AA2 represents —CH₂—CH(R₁)—CH₂— or —CH₂—CH(R₁)—CO—whereupon R₁ has the same meaning as defined above.

AA3 represents:

wherein m is an integer of 1 or more, and R₁, R₁′ and R₂ have the samemeanings as defined above, provided that when m is an integer of 2 ormore, the two substituent groups R₁ may be the same or different; thisalso applies to R₁′ and R₂.

The saturated or unsaturated alkyl containing one or more carbon atoms,which is represented by X, is preferably C₁₋₂₀ alkyl such as methyl,ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-heptyl, n-hexyl,n-decyl, vinyl, propanyl or hexenyl.

The acyl represented by X includes C₂₋₁₀ carboxylic acid acyl such asformyl, acetyl, propionyl or benzoyl; or C₇₋₁₃ sulfonic acid acyl suchas benzenesulfonyl naphthalene sulfonyl or the like.

The group represented by R₁ or R₁′ is preferably a group represented bye.g. formula (2):

—(CH₂)_(n)-P-Q  (2)

(wherein n is an integer of 0 to 10, P is —CO—O—, —O—CO—, —O—, —CO—S—,—CS—S—, —S—CO—, —S—, —CO—NH—, —NH—CO— or —CO—NH—CO—, Q is H or C₁₋₂₀,alkyl represented by X described above.) Further, P may also be —CO—.

In addition, P may be —S—S— or —NH—CS—. In every —NH— described above, Hmay be replaced by a C₁₋₂₅ saturated or unsaturated alkyl group, a C₆₋₂₀aryl group, or a C₇₋₂₃ aralkyl group.

More preferably, P is:

—CO—O—, —CO—, —O—, —S—, —S—S—, —CO—S—, —CO—NH—, —NH—CO— or —NH—CS—.

The group represented by R₁ or R₁′ may be a group having Q bound to—(CH₂)_(n) directly not via P.

It is preferable that the lower alkyl group represented by Z, R6 or R7is C₂₋₆ alkyl such as methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, i-butyl, n-pentyl or n-hexyl.

Hereinafter, preferable embodiments of the peptide compounds accordingto the present invention are described.

(1) Preferable embodiments of AA1: (A) amino acids or peptides such asSer, Gly-Ser or —NH—(CH₂)₃CH(CH₂OH)CO— where a peptide linkage moietybetween two amino acid residues is —(CH₂)₂— and (B) primary amines, forexample —NH— (CH₂)₃CH(CH₂OH)CH₂— where a peptide linkage moiety betweentwo amino acids is —(CH₂)₂—; —NH— (CH₂)₃CH(R₁)CH₂— where a peptidelinkage moiety between two amino acids is —(CH₂)₂—, wherein R₁, has thesame meanings as defined above; and —NH—CH(CH₂OH)CH₂—.

As (A) amino acids or peptides, NH₂—(CH₂)₄—COOH, NH₂—C(CH₃)₂—(CH₂)₃—COOHand NH₂—CH(CH₃)—(CH₂)₂—CH(CH₃)—COOH— can also be exemplified

(2) Preferable embodiments of AA2: (A) amino acids such as Ser, homoSer,Cys, homoCys, Asp, Glu, Lys, Ala, Val, Leu, homoLeu, Ile, homoIle,ornithine, aminoadipic acid, methionine, ethionine, butionine, andS-methyl cysteine, among which Ser is particularly preferable, and (B)structures other than amino acid residues; for example, there can bementioned —CH₂—CH(R₁)—CO—, —CH₂—CH(R₃)—CH₂—, etc. wherein R₁ has thesame meanings as defined above.

In particular, amino acids (a) with a hydrophobic side chain, such asleucine, valine, norleucine, homoleucine, homoisoleucine, naphthylalanine or its analogues thereof, tryptophan, phenylalanine,cyclohexylalanine etc. or N-methylamino acids thereof are preferable.Further, amino acids (b) with a side chain having a functional groupcapable of modification with acyl group, alkyl group, alkenyl group oraralkyl group, such as serine, homoserine, threonine, cysteine,homocysteine, aspartic acid, glutamic acid, adipic acid, lysine,ornithine etc. and N-methylamino acids thereof are preferable.

An acyl group, alkyl group, alkenyl group or aralkyl group etc. arebound to side chains of the amino acids (b) via an ester, amide,disulfide, ether, thioether, thioester, carbamide or thiocarbamidelinkage. Further, an alkyl or aralkyl group may be bound to the α-carbonof the amino acid.

(3) Preferable embodiments of AA3: Amino acids or peptides such as Pheor a peptide having an amino acid sequence of from Phe at the 4thposition to Arg at the 28th position from the amino-terminal in theamino acid sequence set forth in SEQ ID NO: 2 or 3, or peptides havingan amino acid sequence wherein in the amino acid sequence set forth inSEQ ID NO: 2 or 3, one amino acid is sequentially deleted starting fromthe carboxyl-terminal amino acid until Leu at the 5th position from theamino-terminal. For example, AA3 includes:

Phe Leu, Phe Leu Ser, Phe Leu Ser Pro, Phe Leu Ser Pro Glu,Phe Leu Ser Pro Glu His,  Phe Leu Ser Pro Glu His Gln,Phe Leu Ser Pro Glu His Gln Arg, Phe Leu Ser Pro Glu His Gln Lys Ala,Phe Leu Ser Pro Glu His Gln Lys Ala Gln,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser Lys,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser Lys Lys,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser Lys Lys Pro,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser LysLys Pro Pro,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser LysLys Pro Pro Ala,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser LysLys Pro Pro Ala Lys,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser LysLys Pro Pro Ala Lys Leu,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser LysLys Pro Pro Ala Lys Leu Gln,Phe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu SerLys Lys Pro Pro Ala Lys Leu Gln Pro, andPhe Leu Ser Pro Glu His Gln Lys Ala Gln Gln Arg Lys Glu Ser LysLys Pro Pro Ala Lys Leu Gln Pro Arg.

As a matter of course, the amino acids exemplified as AA3 may be L-aminoacids or D-amino acids. Further, in the amino acid sequences exemplifiedas AA3, one to several amino acids (preferably up to about ⅓ of theamino acid sequence) may be replaced by non-natural amino acid units ornon-amino acid units, for example:

-   -   —NH—(CH₂)₃CH(CH₂OH)—    -   —NH—(CH₂)₃CH(CH₂OH)CO—    -   —NH—CH(CH₂OH)CH₂—    -   —NH—(CH₂)₃CH(R₁)CH₂—    -   —CH₂—CH(R₁)—CO—, or    -   —CH₂—CH(R₁)—CH₂—        wherein R₁ has the same meanings as defined above. When AA3        contains a plurality of groups represented by the above formulae        and a plurality of groups represented by R₁, these groups are        the same or different.

Further, any amino acids exemplified as AA3 may have substituent groupsrepresented by R₁ described above. When a plurality of R₁ groups arepresent in a group represented by AA3, these R₁ groups may be the sameor different.

When amino acids constituting the peptide have hydroxyl group, mercaptogroup, imino group or amino group in their side chains, preferableexamples of such side chains are shown below. In the following examples,R8 is a saturated or unsaturated alkyl group containing one or morecarbon atoms. Such alkyl chain may have the same meanings as defined forthe above-described alkyl chain shown by X.

A) Side chain of Ser; —CH₂—O—CO—R8 or —CH₂—O—R8,

B) Side chain of homoSer; —CH₂—CH₂—O—CO—R8 or —CH₂—CH₂—O—R8,

C) Side chain of Cys; —CH₂—S—CO—R8 or —CH₂—S—R8,D) Side chain of homoCys; —CH₂—CH₂—S—CO—R8 or —CH₂—CH₂—S—R8,E) Side chain of Asp; —CH₂—COO—R8 or —CH₂—CO—NH—R8,F) Side chain of Glu; —CH₂—CH₂—COO—R8 or —CH₂—CH₂—CO—NH—R8G) Side chain of Lys; —(CH₂)₄—NH—CO—R8,H) Side chain of aminoadipic acid; —CH₂—CH₂—CH₂—COO—R8 orCH₂—CH₂—CH₂—CO—NH—R8,I) Side chain of ornithine; —(CH₂)₃—NH—CO—R8.J) An alkyl side-chain in an amino acid such as Ala, Val, Leu,homoleucine, Ile, homoisoleucine, S-methyl cysteine, methionine,ethionine, or butionine maybe a modified alkyl group shown in theformula (2) as described above.

Further, the present invention encompasses, as preferable embodiments,an agent for increasing the intracellular calcium ion concentration oran agent for inducing GH secretion, which comprises a partial peptideconsisting of amino acids of from the amino-terminal to 13th, 14th or15th position in the amino acid sequence of SEQ ID NO: 2 or 3. In thiscase, it is not always necessary that the respective amino acid unitsconstituting the partial peptide are chemically modified.

Further, a preferable embodiment of the present invention is thefollowing peptide-type compound.

A ghrelin derivative refers to a peptide-type compound wherein thechemical structure of natural ghrelin is partially modified, andshort-chain ghrelin refers to a peptide consisting of less than 27 or 28amino acids, which is derived from natural ghrelin of 27 to 28 aminoacids by deleting some of the amino acids. Further, an amino acidresidue at the n-position refers to an amino acid residue at then-position from the amino-terminal.

The amino-terminal amino acid of ghrelin or its short-chain ghrelinderivative may be any amino acid (the amino-terminal amino acid ofnatural ghrelin is glycine) insofar as the α-amino group of said aminoacid is not protected, or may be either D- or L-amino acid, but it ispreferably alanine, valine, aminoisobutanoic acid or butanoic acid.

The 2nd residue may be any amino acid (e.g. serine in the naturalghrelin), preferably an amino acid having a small side-chain, such asalanine, serine, histidine, norvaline or a non-amino acid compound.

The 1st and 2nd residues may be a δ-amino acid corresponding to twoamino acids, for example, 5-aminopentanoic acid,5-amino-5-dimethylpentanoic acid, 2,5-diaminopentanoic acid etc.exemplified in the Examples.

The amino acid residues selected at the 3rd and 4th positions may be D-or L-amino acids, D- or L-N-methylamino acids, or a combination of theseamino acids. In particular, it is preferable that the amino acid at the3rd positions is an L-amino acid or both the amino acids at the 3rd and4th positions are L-amino acids.

The steric configuration of the amino acid residues selected at the 3rdand 4th positions can be suitably selected depending on the sequence ofamino acids at 1st and 2nd positions. That is, both the 3rd and 4thpositions residues are preferably L-amino acids in the case of the aminoacid sequence Gly-Ser at the 1st and 2nd positions in natural ghrelin,whereas both the 3rd and 4th positions residues may be D-amino acids inthe case of another amino acid sequence such as Aib-His. Further, if theresidues at the 1st and 2nd positions are an δ-amino acid (e.g.,aminopentanoic acid) having a length of 2 amino acids, the residues atthe 3rd and 4th positions may be L- or D-amino acids.

The amino acid residues selected at the 3rd and 4th positions arepreferably D- or L-amino acids such as leucine, valine, norleucine,homoleucine, homoisoleucine, naphthyl alanine and its homologuesthereof, tryptophan, phenylalanine and cyclohexyl alanine or D- orL-N-methylamino acids thereof.

The amino acid residues selected at the 3rd and 4th positions are morepreferably aromatic hydrophobic amino acids such as naphthyl alanine andits homologues thereof, tryptophan, phenylalanine and cyclohexylalanine, among the hydrophobic amino acids described above.

Further, the amino acid residues selected at the 3rd and 4th positionsare preferably basic amino acids such as lysine, arginine and histidine.Especially, lysine is preferable.

The ghrelin molecule is to be basic by these basic amino acids, thusfurther improving the Ca-releasing activity.

The amino acid residues selected at the 3rd and 4th positions arepreferably those having functional groups in their side chains, whichcan be modified with an acyl group (alkanyl group, alkenonyl group oraryl alkanyl group), an alkyl group or an aralkyl group, and preferableexamples of such amino acids residues include serine, homoserine,threonine, cysteine, homocysteine, aspartic acid, glutamic acid, adipicacid, lysine, ornithine etc.

The amino acids having these reactive side chains may be either D- orL-amino acids or their corresponding D- or L-N-methyl amino acids, butit is particularly preferable that the 3rd position residue is anL-amino acid or both the 3rd and 4th positions residues are L-aminoacids.

Further, to side chains of these amino acids may be bound an acyl groupsuch as alkanyl group (the number of carbon atoms thereof is 2 to 35,preferably 6 to 18, more preferably 8 to 12), an alkenonyl group (thenumber of carbon atoms thereof is 2 to 35, preferably 6 to 18, morepreferably 8 to 12), an aryl alkanyl group (benzoyl, phenacetyl, phenylbutyryl, naphthoyl, naphthyl acetyl or naphthyl propionyl group etc.),an alkyl group (the number of carbon atoms thereof is 2 to 35,preferably 6 to 18, more preferably 8 to 12), or an aralkyl group(benzyl, phenetyl, phenyl propyl, phenyl butyl, phenyl pentyl, naphthylmethyl group etc.) via a carbamate, thiocarbamate, ester, amide,disulfide, ether, thioether or thioester linkage. Further, theaforementioned alkyl and aralkyl groups may be bound not via the linkageto the α-carbon atoms of amino acids at the 3rd and 4th positions.

The combination of amino acid residues selected at the 3rd and 4thpositions is preferably a combination of an amino acid having ahydrophobic side chain as the 3rd position amino acid residue and ahydrophobic amino acid as the 4th position amino acid residue.

The 3rd position amino acid residue having a hydrophobic side chain ispreferably a modified amino acid into the α carbon of which (a) asaturated or unsaturated alkyl chain containing one or more carbon atomswas introduced via or not via an alkylene group containing one or morecarbon atoms and via an ester, ether, thioether, amide or disulfidelinkage, or (b) a saturated or unsaturated alkyl chain containing one ormore carbon atoms was introduced. In particular, a modified amino acidinto the α-carbon of which a saturated alkyl chain containing one ormore carbon atoms was introduced is more preferable.

The carboxyl group of amino acid at the 4th position may be an amide, analkyl amide (e.g. methyl amide or ethyl amide), a benzyl amide, or anaralkyl amide (e.g. adamantane amide or adamantane alkyl amide).

Further, a basic group such as amino group or guanidido group may bebound to the alkyl or aralkyl amide. The basic group includes e.g.—CONH—CH₂CH₂—NH₂, —CONH—CH₂NHCH₃, —CONH—CH₂CH₂CH₂—NH—C(NH₂)═NH, and—CONHCH₂Ph-NH₂.

A basic amino acid such as arginine, lysine and histidine maybe added tothe carboxyl group of amino acid at the 4th position, and this basicamino acid may be a D- or L-amino acid, a racemate, or D- or L-N-methylamino acid.

The carboxyl group of the amino acid may be an alkyl or aralkyl amide asdescribed above. Further, a basic group such as amino group or guanididogroup may be added to the alkyl or aralkyl amide. The basic groupincludes those exemplified above.

As an amino acid sequence of amino acid at the 5th position andsubsequent amino acids, a sequence of any length consisting of leucineat the 5th position and subsequent amino acids up to amino acid at the28th position in human or rat ghrelin may be added to amino acid at the4th position.

Such amino acid sequences are preferably ghrelin (1-5), ghrelin (1-6),ghrelin (1-7), ghrelin (1-8), ghrelin (1-9), ghrelin (1-10), and ghrelin(1-11) where ghrelin (m-n) refers to a peptide having an amino acidsequence at the m- to n-positions from the amino-terminal in ghrelin. Inparticular, ghrelin (1-5) is preferable.

The carboxyl-terminal thereof is preferably an alkyl or aralkyl amide asdescribed above.

Further, a basic group such as amino group or guanidido group may bebound to the alkyl or aralkyl amide. The basic group includes thoseexemplified above.

Further, a basic amino acid such as arginine, lysine and histidine maybe added to the carboxyl-terminal amino acid of acarboxyl-terminal-deleted ghrelin derivative wherein an amino acidsequence of any length consisting of amino acid 5 and subsequent aminoacids up to amino acid 28 was added to the carboxyl-terminal of ghrelin(1-4).

This basic amino acid may be a D- or L-amino acid, a racemate, or D- orL-N-methyl amino acid.

The carboxyl group of the basic amino acid may be an alkyl or aralkylamide as described above. Further, a basic group such as amino group orguanidido group may be bound to the alkyl or aralkyl amide. The basicgroup includes those exemplified above.

In a particularly preferable embodiment, the carboxyl-terminal aminoacid of ghrelin (1-5), ghrelin (1-6) and ghrelin (1-7) is a D- orL-amino acid or its corresponding D- or L-N-methyl amino acid.

Further, basic amino acids such as arginine, lysine and histidine maybeadded to resides at the 5th, 6th and 7th position, and these basic aminoacids maybe D- or L-amino acids, racemates, or D- or L-N-methyl aminoacids.

The carboxyl group of such a basic amino acid may be an alkyl or aralkylamide as described above. Further, a basic group such as amino group orguanidido group may be bound to the alkyl or aralkyl amide. The basicgroup includes those exemplified above.

In a preferable embodiment in the present invention, the peptidecompound of the present invention in the case where thecarboxyl-terminal is an alkyl or aralkyl amide as described above may bean amide derivative wherein an amino group is further bound to the alkylor aralkyl group. Specifically, a peptide compound wherein thecarboxyl-terminal is e.g. aminoethyl amide can be mentioned.

The peptide-type compound of the present invention wherein thecarboxyl-terminal is an amide or an amide derivative as described aboveis a useful compound because of its resistance to decomposition byenzymes such as carboxy peptidases in vivo.

Similarly, the peptide-type compound of the present invention includingN-methyl amino acid is also a useful compound because of its resistanceto enzymes.

The peptide-type compound of the present invention can be obtained in ausual manner. For example, it can be isolated from a natural source orproduced by recombinant DNA technology and/or chemical synthesis.Further, when a modification (e.g., acylation) in the amino acidresidues is necessary, the peptide compound can be subjected to amodification reaction by well-known methods in the art.

Specifically, the peptide-type compound of the present invention can beobtained by culturing host cells transformed with an expression vectorharboring a DNA coding the peptide of the present invention and thenrecovering the desired peptide from the culture.

By selecting the host cells, a compound having the desired peptidemodified by e.g. acylation in the cells can be obtained. When saidpeptide is not modified, a modification reaction such as acylation canbe conducted as necessary by well-known methods in the art. For theacylation reaction, enzymes such as lipase can also be used.

The vector in which the gene is to be integrated includes e.g. E. colivectors (pBR322, pUC18, pUC19 etc.), Bacillus subtilis vectors (pUB110,pTP5, pC194 etc.), yeast vectors (YEp type, YRp type, YIp type), oranimal cell vectors (retrovirus, vaccinia virus etc.), but any othervectors capable of maintaining the desired gene stably in host cells canalso be used. The vector is introduced into suitable host cells. Forintegrating the desired gene into a plasmid and introducing the plasmidinto host cells, methods described in Molecular Cloning (Sambrook etal., 1989) can be used.

To express the desired peptide gene in the above plasmid, a promoter islinked operatively upstream of said gene.

The promoter used in the present invention may be any suitable promotercompatible with host cells used for expression of the desired gene. Forexample, lac promoter, trp promoter, lpp promoter, XPL promoter, recApromoter etc. can be used in the genus Escherichia as host cells to betransformed; SP01 promoter, SP02 promoter etc. can be used in the genusBacillus; GAP promoter, PH05 promoter, ADH promoter etc. can be used inyeasts; and SV40-derived promoter, retrovirus-derived promoter etc. canbe used in animal cells.

The desired gene-containing vector obtained in this manner is used totransform host cells. The host cells include microorganisms (forexample, the genus Escherichia, the genus Bacillus etc.), yeast (thegenus Saccharomyces, the genus Pichia, the genus Candida etc.), animalcells (CHO cells, COS cells etc.) etc. The medium for culture ispreferably a liquid medium, and particularly preferably the mediumcontaining a carbon source, a nitrogen source etc. necessary for growthof the transformed cells to be cultured. If desired, vitamins, growthpromoters, serum etc. can be added.

For directly producing the fatty acid-modified peptide, the cells arepreferably those having the activity of a processing protease capable ofcutting a suitable site in a precursor polypeptide of said peptide andthe activity of acylating the serine residue in said peptide. Host cellshaving such processing protease activity and serine-acylating activitycan be obtained by transforming host cells with an expression vectorcontaining a cDNA coding said precursor polypeptide and then selectingthe transformed cells by confirming whether or not they produce thefatty acid-modified peptide having a Ca-releasing activity or a GHsecretion-inducing activity.

After culture, the peptide of the present invention is separated andpurified from the culture in a usual manner. To extract the desiredproduct from the cultured microorganisms or cells, for example, themicroorganisms or cells after culture are collected and suspended in abuffer containing a protein denaturant (e.g. guanidine hydrochloride)and the microorganisms or cells are disrupted by sonication etc. andthen centrifuged. To purify the desired product from the supernatant,separation and purification methods such as gel filtration,ultrafiltration, dialysis, SDS-PAGE, various chromatographic techniquescan be suitably combined in consideration of the molecular weight,solubility, charge (isoelectric point), affinity etc. of the desiredproduct.

The peptide compound of the present invention can be chemicallysynthesized in a usual manner. For example, amino acids havingprotective groups are condensed by a liquid phase method and/or a solidphase method to extend a peptide chain, then all protective groups areremoved therefrom by an acid, and the resulting crude product ispurified by the above purification techniques to give the desiredpeptide compound. An amino acid residue at the desired site can beacylated selectively by an acylase or acyl transferase.

Various methods have been well established for production of peptides,and the peptide-type compound of the present invention can also beeasily produced by such known methods. For example, the peptide-typecompound may be synthesized by the classical peptide synthesis method orby the solid phase method.

Hereinafter, a process for producing the peptide compound of the presentinvention by a combination of recombinant DNA technology and chemicalsynthesis is described by reference to examples.

Active esters of amino-terminal peptides, for example, (1)Boc-Gly-Ser(Bu)-Ser(R10)-Osu, (2) Boc-Gly-Ser(Bu)-Ser(R10)-Phe-Osu, and(3) Boc-Gly-Ser(Bu)-Ser(R10)-Phe-Leu-Osu, are chemically synthesized andthen bound to carboxyl-terminal peptides produced by recombinant DNAtechnology, that is, (4) FLSPEHQRVQQRKESKKPPAKLQPR, (5)LSPEHQRVQQRKESKKPPAKLQPR, and (6) SPEHQRVQQRKESKKPPAKLQPR, respectively;that is, (1) is bound to (4), (2) to (5), and (3) to (6), wherebypeptide compounds each consisting of 28 amino acids are obtainedrespectively. Specifically, XXXXZSPEHQRVQQRKESKKPPAKLQPR is expressed inE. coli followed by protection of its amino groups with Boc2(O) to giveBoc-XXXXZSPEHQRVQQRK(Boc)ESK(Boc)K(Boc)PPAK(Boc)LQPR. Then, theresulting peptide is converted intoNH₂-SPEHQRVQQRK(Boc)ESK(Boc)K(Boc)PPAK(Boc)LQPR by cleavage with anenzyme selective for the carboxyl-terminal of amino acid Z. Thiscompound is mixed with Boc-Gly-Ser(Bu)-Ser(R10)-Phe-Leu-Osu in anaqueous neutral to weak alkaline solution, and the resultingBocGlySer(Bu)Ser(R10)FLSPEHQRVQQRK(Boc)ESK(Boc)K(Boc)PPAK(Boc)LQPR istreated with trifluoroacetic acid, whereby the desired product can beobtained.

The above one-letter notation of amino acid is in accordance with adescription in Cellular Molecular Biology, 3rd edition published on Dec.10, 1997 by Newton Press Co., Ltd.

In addition, Boc represents t-butyloxycarbonyl, Osu represents a residuederived from N-hydroxysuccinimide by eliminating hydrogen from thehydroxyl group thereof, Bu represents a butyl group, and R10 representsthe substituent group of the modified amino acid according to thepresent invention.

Salts of the peptide-type compound of the present invention arepreferably pharmaceutically acceptable salts including, for example,salts with inorganic bases, salts with organic bases, salts withinorganic acids, salts with organic acids, and salts with basic oracidic amino acids.

Preferable examples of the salts with inorganic bases include alkalimetal salts such as sodium salts, potassium salts etc.; alkaline earthmetal salts such as calcium salts, magnesium salts etc.; and aluminumsalts, ammonium salts etc.

Preferable examples of the salts with organic bases include salts withtrimethylamine, triethylamine, pyridine, picoline, ethanolamine,diethanolamine, triethanolamine, dicyclohexyl amine,N,N′-dibenzylethylene diamine etc.

Preferable examples of the salts with inorganic acids include salts withhydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,phosphoric acid etc.

Preferable examples of the salts with organic acids include salts withformic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalicacid, tartaric acid, maleic acid, citric acid, succinic acid, malicacid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acidetc.

Preferable examples of the salts with basic amino acids include saltswith arginine, lysine, ornithine etc., and suitable examples of thesalts with acidic amino acids include salts with aspartic acid, glutamicacid etc.

Among these salts, sodium salts and potassium salts are most preferable.

The peptide-type compound of the present invention or pharmaceuticallyacceptable salts thereof are low toxic and have a GH-secretion inducingaction, and they can be administered as such or after mixed with known,pharmaceutically acceptable carriers, excipients, vehicles augmentorsetc., to a mammal (for example, human being, mouse, rat, rabbit, dog,cat, bovine, horse, porcine, monkey etc.). In the case of intravenousinjection into an adult, the daily dose is 0.01 to 5 mg/kg, preferably0.04 to 1.5 mg/kg. This dose is administered desirably once to thriceevery day. The peptide-type compound of the present invention iscompounded with pharmaceutically acceptable carriers and can be orallyor parenterally as solid pharmaceutical preparations such as tablets,capsules, granules, powders etc. or as liquid pharmaceuticalpreparations such as syrups, injections etc.

The pharmaceutically acceptable carriers include a wide variety oforganic or inorganic carriers which are customarily used aspharmaceutical materials, and these are compounded as vehicles,lubricants, binders, disintegrant in solid pharmaceutical preparationsor as solvents, adjuvant, suspending agents, isotonicity-conferringagents, buffers and soothing agents in liquid pharmaceuticalpreparations.

As necessary, pharmaceutical additives such as preservatives,antioxidants, coloring agents, sweeteners etc. can also be used.

Preferable examples of the vehicles include e.g. lactose, white sugar,D-mannitol, starch, crystalline cellulose, light anhydrous silicic acidetc. Preferable examples of the lubricants include magnesium stearate,calcium stearate, talc, colloidal silica etc.

Preferable examples of the binders include crystalline cellulose, whitesugar, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinyl pyrrolidone etc.

Preferable examples of the disintegrant include starch, carboxymethylcellulose, carboxymethyl cellulose calcium, croscalomelose sodium,carboxymethyl starch sodium etc.

Preferable examples of the solvents include water for injection,alcohol, propylene glycol, macrogol, sesame oil, corn oil etc.

Preferable examples of the solubilizers include polyethylene glycol,propylene glycol, D-mannitol, benzyl benzoate, ethanol,trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodiumcitrate etc.

Preferable examples of the suspending agents include surfactants such asstearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionicacid, lecithin, benzalconium chloride, benzethonium chloride andglycerin monostearate, and hydrophilic polymers such as polyvinylalcohol, polyvinyl pyrrolidone, carboxymethyl cellulose sodium, methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose andhydroxypropyl cellulose.

Preferable examples of the isotonicity-conferring agents include sodiumchloride, glycerine, D-mannitol etc.

Preferable examples of the buffers include buffer solutions such asphosphates, acetates, carbonates, citrates etc.

Preferable examples of the soothing agents include benzyl alcohol etc.

Preferable examples of the preservatives include p-oxyesterbenzoates,chlorobutanol, benzyl alcohol, phenetyl alcohol, dehydroacetic acid,sorbic acid etc.

Preferable examples of the antioxidants include sulfites, ascorbic acidetc.

The above pharmaceutical composition brings about an effect equal to orhigher than the effect of GH upon administration and can reduce variousside effects caused by administration of GH.

As diseases attributable to deficiency or reduction in GH, the diseasesto which the pharmaceutical composition can be applied or the effects ofthe pharmaceutical composition include, but are not limited to,activation of osteoblasts and re-constitution of bone in people withdwarfism and normal human beings, enhancement of muscular strength andmuscular amount in GH-deficient adults, improvement of motility inGH-deficient adults, remedy of heavy burns in children, its combined usewith gonadotropins in induction of ovulation, prevention ofabnormalities in protein metabolism by administration of prednisone,promotion of T cell “education” in heavy immune disorder, the effect ofinhibiting reduction of the body weight of the aged and the effect ofenlarging adipose tissues and preventing dermal atrophy.

Further, the diseases or effects not directly correlated with deficiencyor reduction in GH include e.g. the effect of increasing pulsatile flowas shown in Example 7, and thus it is effective for treatment of cardiacdiseases such as cardiac failure etc.

The effect of the pharmaceutical composition is not restricted to humanbeings. That is, it has an effect on growth promotion for animals,reduction of fat in meat, etc., which is equal to or higher thanadministered GH.

For example, as shown in Example 13, the pharmaceutical composition ofthe present invention exhibits an appetite-promoting action uponadministration into ventricle or intravenous administration, so it canbe used as an appetite promoter for treating anorexia or sitophobia.

In addition, as shown in Example 14, the pharmaceutical composition ofthe present invention has a stomach motility- and gastric acidsecretion-promoting action, and thus it can also be used as an agent fortreating stomach functional diseases such as non-diabrotic dyspepsia,sudden light stomach atony, functional dyspepsia, and refluxesophagitis.

Further, as shown for example in Example 15, the pharmaceuticalcomposition of the present invention exhibits a cell growth-promotingaction in bone marrow, duodenum and jejunum by intravenousadministration, and thus it can be used as an agent for protecting smallintestine tunica mucosa, an agent for preventing damage to smallintestine tunica mucosa during intravenous nutrition and an agent fortreating osteoporosis.

Further, the pharmaceutical composition described above has an effectfor treating the diseases or for improving the physical conditionsdescribed below.

For example, it can be used for stimulative treatment for release ofgrowth hormones in the aged, prevention of catabolic side effects ofsugar corticoids, prevention and treatment of osteoporosis, stimulationat immune system, promotion of remedy of damages, promotion of repair ofbroken bone, treatment of growth delay, treatment of renal insufficiencyor functional insufficiency attributable to growth delay, treatment ofinsufficient conditions correlated with physiological insufficientconditions including growth hormone-deficient children and chronicdiseases, treatment of obesity and growth delay correlated with obesity,treatment of growth delay correlated with Plauda-Villi syndrome andTaner syndrome, promotion of recovery of burn patient and reduction inhospitalization, treatment of intrauterine growth delay, skeletonmulformation, hypercorticoid disease and Cusshing syndrome, induction ofrelease of pulsatile growth hormone, replacement of growth hormone instressed patients, cartilage mulformation, Noonan syndrome,schizophrenia, depression, Alzheimer's disease, remedy of delayed repairof damage and physicosocial deprivation, treatment of pulmonaryinsufficiency and respiratory organ dependence, decrease of catabolicreaction of protein after major operation, reduction of protein loss andcachexia caused by chronic diseases such as cancer and AIDS, treatmentof hyperinsulinism including pancreas nesidioblastosis, adjuvant therapyfor induction of ovulation, and treatment of patients with immunerepression, improvement of muscular strength and motility, maintenanceof thickness of skin in the aged, metabolic homeostasis and renalhomeostasis, stimulation of osteoblast, re-formation of bone andstimulation of cartilage growth, in order to stimulate growth of thymusand to prevent deterioration in thymic functions accompanying aging.

Further, the following effects on animals are also expected. Forexample, mention is made of an increase in the rate of animal growth, anincrease in production of milk and fur in animals, stimulation of theimmune system in pet animals, treatment of diseases caused by advancedage in pet animals, growth promotion of domestic animals, and anincrease in fur in sheep.

An antibody whose antigen is fatty acid-modified peptide of the presentinvention having a Ca-releasing activity or a GH secretion-inducingactivity can be obtained by a method known in the art. The antibody maybe a monoclonal or polyclonal antibody, and can be obtained by a methodknown in the art. Further, a method for measuring the fattyacid-modified peptide using said antibody and a measuring kit using saidmeasuring method can also make use of a method known in the art. Asdescribed in Example 17, antibodies to amino- and carboxyl-terminalpeptides from ghrelin are prepared respectively, and since the formerrecognizes fatty acid-modified serine at the 3rd position, both theantibodies can be used to separate and quantify ghrelin modified with afatty acid and ghrelin from which the fatty acid was eliminated.

The antibodies to amino- and carboxyl-terminal peptides in ghrelin canbe obtained in a known method, and they may be monoclonal or polyclonalantibodies.

For the present peptide-type compound having a modified amino acid atthe 3rd position from the amino-terminal or a pharmaceuticallyacceptable salt thereof, an antibody which specifically recognizes aside chain of 3rd amino acid residue (preferably a fatty acid) and bindsto an amino-terminal partial peptide of the peptide-type compound canalso be produced in the same manner. In addition, for the peptide-typecompound of the present invention or a pharmaceutically acceptable saltthereof, an antibody which binds specifically to the peptide having amodified amino acid can also be produced in the same manner.

The present invention also encompasses an examination kit comprising acombination of an antibody specifically recognizing a side chain of themodified amino acid and an antibody recognizing amino acids (or apeptide) excluding the modified amino acid and/or a non-amino acidcompound, preferably an antibody to a carboxyl-terminal partial peptideof the peptide-type compound of the present invention or of apharmaceutically acceptable salt thereof, as described above.

Further, the present invention encompasses an assay method wherein thepeptide-type compound of the present invention having a modified aminoacid, preferably an acylated amino acid, or a pharmaceuticallyacceptable salt thereof, and the peptide-type compound of the presentinvention not containing a modif ied amino acid, or a pharmaceuticallyacceptable salt thereof, are separated and detected by use of saidexamination kit.

Hereinafter, the assay method and examination kit described above aredescribed by reference to their embodiments, which however are notintended to limit the present invention.

That is, the assay method includes, for example, (i) a method ofquantifying the peptide-type compound etc. of the present invention in atest solution, which comprises allowing a test material in a testsolution and the labeled peptide-type compound etc. of the presentinvention to react competitively with an antibody to the peptide-typecompound etc. of the present invention and then determining the ratio ofthe labeled peptide-type compound etc. of the present invention bound tosaid antibody, and (ii) a method of quantifying the proteins etc. of thepresent invention in a test solution, which comprises allowing a testsolution to react with the antibody of the present inventioninsolubilized on a carrier and another labeled antibody of the presentinvention simultaneously or successively and then measuring the activityof the labeling agent on the insolubilizing carrier and/or the activityof the labeling agent not captured on the insolubilizing carrier. In thequantification methods (i) and (ii), it is preferable that one antibodyis an antibody recognizing an amino-terminal region of the protein etc.of the present invention, while the other antibody is an antibodyreacting to a carboxyl-terminal region of the protein etc. of thepresent invention.

In the method of assaying the peptide-type compound etc. of the presentinvention, the monoclonal antibody against said compound (also referredhereinafter to as the anti-protein antibody) can be used not only forquantifying the protein etc. of the present invention, but also fordetection thereof by tissue staining etc.

For these purposes, either the antibody molecule itself or an F(ab′)₂,Fab′ or Fab fraction of the antibody molecule may be used.

The method of quantifying the peptide-type compound etc. of the presentinvention by use of said antibody is not particularly limited as long asthe method comprises detecting the amount of the antibody correspondingto the amount of the antigen (e.g., the amount of the protein), theantigen or an antigen-antibody conjugate in a test solution, by chemicalor physical means and calculating it on the basis of a standard curveprepared using standard solutions containing known amounts of theantigen. For example, nephelometry, the competitive method, theimmunometric method and the sandwich method are preferably used, but thesandwich method described later is particularly preferably used inrespect of sensitivity and specificity.

For a measurement method using a label in the assay method of thepresent invention, the label includes e.g. a radioisotope, an enzyme, afluorescent material and a luminescent material.

The radioisotope includes, for example, ¹²⁵I, ¹³¹I, ³H, ¹⁴C etc.

The enzyme is preferably a stable enzyme with high specific activity,which includes, for example, β-galactosidase, β-glucosidase, alkaliphosphatase, peroxidase and malate dehydrogenase.

The fluorescent material includes, for example, fluorescamine andfluorescein isocyanate.

The luminescent material includes, for example, luminol, luminolderivatives, luciferin, and lucigenin.

Further, a biotin-avidin system can also be used for binding the labelto the antibody or antigen.

Hereinafter, the present invention is described in more detail byreference to the Examples. Unless otherwise specified, the geneticmanipulation means were in accordance with Molecular Cloning (Sambrooket al., 1989).

Example 1 Creation of a Cell Strain Expressing GHS-R and Measurement ofCa-Releasing Activity

To assay an increase in the intracellular calcium ion concentration(Ca-releasing activity) occurring upon binding of a GH secretagogue(GHS) to GHS-receptor (GHS-R), a cell strain expressing rat GHS-R wascreated in the following manner. A full-length cDNA for rat GHS-R wasobtained by RT-PCR (reverse

Example 1 Creation of a Cell Strain Expressing GHS-R And Measurement ofCa-Releasing Activity

To assay an increase in the intracellular calcium ion concentration(Ca-releasing activity) occurring upon binding of a GH secretagogue(GHS) to GHS-receptor (GHS-R), a cell strain expressing rat GHS-R wascreated in the following manner. A full-length cDNA for rat GHS-R wasobtained by RT-PCR (reverse transcriptase-polymerase chain reaction)where a cDNA derived from rat brain was used as template. From thenucleotide sequence of known rat GHS-R [K. K. Mckee, et al, MolecularEndocrinology 11, 415-423 (1997)], sense and antisense primersconsisting of the following nucleotide sequences were synthesized.

Sense primer: 5′-ATGTGGAACGCGACCCCCAGCGA-3′ Antisense primer:5′-ACCCCCAATTGTTTCCAGACCCAT-3′

The amplified cDNA was ligated to vector pcDNAIII (Invitrogen) toconstruct expression vector GHSR-pcDNAIII. CHO cells were transformedvia the expression vector, and the transformed cells stably expressingGHS-R were selected in a medium containing 1 μg/ml G418. The selectedcell strain CHO-GHSR62 responded to 10⁻¹⁰ to 10⁻⁹ M GHRP-6 (GrowthHormone-Releasing hexapeptide). A change in the intracellular calciumion concentration (Ca-releasing activity) was measured by an FLIPRsystem (Molecular Device). Before this measurement, 4×10⁴ CHO-GHSR62cells were put to a 96-well microplate with black wall (Corning Co.,Ltd) and cultured for 12 to 15 hours. Then, the cells were incubatedwith 4 μM fluorescent Fluo4 (Molecular Probe Co., Ltd) for 1 hour andwashed four times with Hank's BSS (Hank's Balanced Salt Solution)containing 20 mM Hepes([N-2-hydroxyethyl]-piperazine-N-[2-ethanesulfonic acid]) and 2.5 mMprobenecid, and the Ca-releasing activity was assayed by adding a sampleand measuring a change in the fluorescence.

Example 2 Purification of an Endogenous GH Secretion-Inducing Peptide

Using the CHO-GHSR62 cells described in Example 1, a wide variety ofrat-derived tissues and organs were examined for their Ca-releasingactivity, and as a result, it was found that a peptide extract derivedfrom rat stomach has a strong Ca-releasing activity even in a smallamount of 5 mg. Accordingly, a peptide having the Ca-releasing activitywas purified from a rat stomach extract by several kinds ofchromatography.

40 g fresh rat stomach was boiled for 5 minutes in 5-fold boiling waterto inactivate proteases present in it. After cooling, the boiled samplewas placed in 1M AcOH-20 mM followed by extracting the peptide by aPolytron mixer. The extract was centrifuged at 11,000 rpm for 30 min.,and the supernatant was concentrated into about 40 ml in an evaporator.The concentrate was precipitated with acetone by adding acetone theretoat a concentration of 66%, and after the formed precipitates wereremoved, the acetone in the supernatant was evaporated. The supernatantwas applied to 10 g Sep-Pak C18 cartridge (Waters Co., Ltd) previouslyequilibrated with 0.1% TFA (trifluoroacetic acid), washed with 10%CH₃CN/0.1% TFA and then eluted with 60% CH₃CN/0.1% TFA. After thesolvent in the eluate was evaporated, the sample was lyophilized. Thelyophilized sample was dissolved in 1 M AcOH and absorbed ontoSP-Sephadex C-25 (H⁺ type) previously equilibrated with 1 M AcOH. Thesample was eluted stepwise with 1 MAcOH, then with 2 M pyridine, andfinally with 2 M pyridine-AcOH (pH 5.0), whereby 3 fractions, that is,SP-I, SP-II and SP-III were obtained respectively. The SP-III fractionwas applied to a Sephadex G-50 gel filtration column, and an aliquot ofeach fraction was assayed for Ca-releasing activity by use of theCHO-GHSR62 cells. A profile in Sephadex G-50 column chromatography isshown in FIG. 1 a, and active fractions (fractions 43 to 48 in FIG. 1 a)having molecular weights of about 3,000 were fractionated by highperformance liquid chromatography (HPLC) by CM-ion exchange on TSKCM-2SW column (4.6×250 mm, Tosoh Corp.) at pH 6.4. The active fractionsby CM-HPLC were secondarily fractionated by CM-HPLC on the same columnat pH 4.8 (FIG. 1 b). The active fractions (elution time of 55 to 56minutes in FIG. 1 b) were purified to homogeneity by reverse phase HPLCon μBondasphere C-18 column (3.9×150 mm, Waters Co., Ltd). From 40 g ofthe rat stomach, 16 μg peptide having a Ca-releasing activity waspurified and designated as ghrelin.

Example 3 Structural Analysis of as Ghrelin

The amino acid sequence of the purified ghrelin derived from rat wasdetermined by a peptide sequencer (ABI 494, Applied Biosysytems Co.,Ltd). The ghrelin was a peptide composed of 28 amino acid residuesconsisting of the following sequence: Gly Ser Xaa Phe Leu Ser Pro GluHis Gln Lys Ala Gln Gln Arg Lys Glu Ser Lys Lys Pro Pro Ala Lys Leu GlnPro Arg (SEQ ID NO: 40) where Xaa is an unidentified amino acid. On thebasis of the nucleotide sequence of the rat cDNA, Xaa was estimated tobe Ser, indicating a certain modification on Ser in the peptide.

Accordingly, unmodified ghrelin wherein serine at the 3rd position fromthe amino-terminal was chemically synthesized by a peptide synthesizer(ABI 433A, Applied Biosystems Co., Ltd). Because the elution time of theunmodified synthetic ghrelin in reverse phase HPLC was significantlydifferent from that of natural ghrelin (FIG. 2 a), the unmodifiedsynthetic ghrelin was found to be more significantly hydrophilic thannatural ghrelin.

From the above result, it was found that in natural ghrelin, serine atthe 3rd position from the amino-terminal (3rd serine) has been modifiedwith a hydrophobic residue.

To reveal the modifying group on 3rd serine, the purified ghrelin wasanalyzed by electrospray ionization mass spectrometry (ESI-MS). Thefound molecular weight (3314.9±0.7) of natural ghrelin was greater by126 than the molecular weight (3188.5) of the unmodified ghrelin peptidewhich was estimated from the nucleotide sequence of the cDNA. From theabove result, it was found that the hydroxyl group of 3rd serine innatural ghrelin has been modified with n-octanoyl (C8:0) fatty acid.

To confirm this, the n-octanoyl (C8:0) ghrelin peptide was chemicallysynthesized and examined for its elution time in reverse phase HPLC. Inchemical synthesis of the n-octanoyl (C8:0) peptide, the peptide whereinall functional groups except for the hydroxyl group of 3rd serine wereprotected was synthesized by the Fmoc solid phase method using a peptidesynthesizer (ABI 433A, Applied Biosystems Co., Ltd), and then thedesired peptide was obtained by acylation of the hydroxyl group of 3rdserine with n-octanoic acid and ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of 4-(dimethylamino)pyridine. The syntheticn-octanoyl peptide indicated the same elution time as that of thepurified natural ghrelin (FIG. 2 a). Further, the synthetic n-octanoylpeptide and a peptide fragment at the 1st to 4th positions from theamino-terminal (Gly 1-Phe 4) which was obtained by treating naturalghrelin with chymotrypsin showed the same retention time in reversephase HPLC.

From the above result, it was concluded that the natural ghrelin derivedfrom rat has the amino acid sequence set forth in SEQ ID NO: 2, whereinthe hydroxyl group of 3rd serine has been acylated with n-octanoic acid(caprylic acid) (FIG. 2 c).

Further, human ghrelin was purified from a human stomach extract, and itwas found that its structure has the amino acid sequence set forth inSEQ ID NO: 3, wherein the side-chain hydroxyl group of 3rd serine hasbeen acylated with n-octanoic acid (caprylic acid) (FIG. 4 a).

The structures of the rat- and human-derived ghrelins were determinedusing those purified as the first-peak fractions (elution time of 55 to56 minutes) out of the active fractions in FIG. 1 b, and afterpurification, the structure of the other active fractions in FIG. 1 bwas also analyzed in the same manner, indicating the presence of notonly caprylic acid (C8:1) but also its monoene acid (C8:1), capric acid(C10:0) and its monoene acid (C10:1), and lauric acid (C12:0) and itsmonoene acid (C12:1) as the modifying fatty acid on 3rd serine.

Further, chicken, eel and frog ghrelins were purified from stomachextracts in the same manner as in Example 2 and analyzed for theirstructure in the same manner as in Example 3. It was found that thechicken ghrelin has the amino acid sequence shown in SEQ ID NO: 25, theeel ghrelin has the amino acid sequence shown in SEQ ID NO: 26, and thefrog ghrelin has the amino acid sequence shown in SEQ ID NO: 27, and inall of the ghrelins, the side-chain hydroxyl group of 3rd serine hasbeen acylated with n-octanoic acid (caprylic acid).

Further, frog (Xenopus Laevis), fish (rainbow trout) and canine ghrelinswere purified from stomach extracts in the same manner as in Example 2and analyzed for their structure in the same manner as in Example 3.

It was found that the frog ghrelin has the amino acid sequence shown inSEQ ID NO: 28, the rainbow trout ghrelin has the amino acid sequenceshown in SEQ ID NO: 29 and 30, and the canine ghrelin has the amino acidsequence shown in SEQ ID NO: 31, and in all of the ghrelins, theside-chain hydroxyl group of 3rd serine or threonine has been acylatedwith n-octanoic acid (caprylic acid).

From the rainbow trout, ghrelin-23 consisting of 23 amino acid residuesshown in SEQ ID NO: 29 and ghrelin-20 consisting of 20 amino acidresidues shown in SEQ ID NO: 30 were obtained.

Example 4 Ca-Releasing Activity of Ghrelin

The natural ghrelin and n-octanoyl-modified synthetic ghrelin had aCa-releasing activity, but the unmodified synthetic ghrelin did not showa significant Ca-releasing activity (FIG. 2 b). Further, becausen-octanoic acid or a mixture of n-octanoic acid and the unmodifiedsynthetic ghrelin did not show a significant Ca-releasing activity, itwas found that the n-octanoic acid residue in the natural ghrelinconstitutes an important structure for the Ca-releasing activity.Hereinafter, ghrelin refers to [O-n-octanoyl-serine 3]-ghrelin (FIG. 2c).

In CHO-GHSR62 cells, the ghrelin exhibited a higher activity ofincreasing the intracellular calcium ion concentration (Ca-releasingactivity) than that by GHRP-6, while GHRH (GH releasing hormone,expressed as GRF in FIG. 3 a) did not exhibit the Ca-releasing activity(FIG. 3 b). The Ca-releasing activity of ghrelin was recognized at aconcentration of 10⁻¹¹ M or more, and its EC₅₀ was 2.5 nM. TheCa-releasing activity of ghrelin was inhibited in the presence of 10⁻⁴ Mspecific antagonist ([D-Lys 3]-GHRP-6) for GHS-R [R. G. Smith, et al.,Science 260, 1640-1643 (1993)], and the Ca-releasing activity wasrestored at a high concentration of ghrelin in the absence of theantagonist (FIG. 3 b). The above result indicates that the Ca-releasingactivity of ghrelin is 10 inhibited antagonistically by the specificantagonist for GHS-R.

Example 5 A cDNA for a Ghrelin Precursor and Expression Thereof inVarious Organs

The amino acid sequence of the ghrelin had no homology with the aminoacid sequences of any known peptides, but as a result of homologyexamination in the GenBank data base, the same sequence was found in arat EST (Expressed Sequence Tag) sequence (GenBank acceptation No.AI549172). On the basis of this EST sequence, the following PCR primerswere synthesized:

(SEQ ID NO: 41) Sense primer: 5′-TTGAGCCCAGAGCACCAGAAA-3′(SEQ ID NO: 42) Antisense primer: 5′ -AGTTGCAGAGGAGGCAGAAGCT-3′.

These 2 primers were used in RT-PCR where a rat stomach-derived cDNA wasused as template. PCR conditions utilized 35 cycles each consisting of98° C. for 10 seconds, 55° C. for 30 seconds and 72° C. for 1 minute.The amplified DNA fragment was used as a probe for screening a ratstomach cDNA library. By screening about 2×10⁵ recombinant phages, afull-length cDNA coding the rat-derived ghrelin was obtained.

The rat ghrelin cDNA was composed of 501 bases shown in SEQ ID NO: 6,coding a ghrelin precursor (prepro-ghrelin) consisting of 117 aminoacids (FIG. 4 a). The amino-terminal 23 amino acid residues in theghrelin precursor had properties as a signal peptide. The ghrelin startsfrom glycine 24, and the last 2 amino acids (Pro-Arg) in the matureghrelin was a sequence undergoing cleavage with a protease.

Using the rat ghrelin cDNA, a human stomach cDNA library was screenedunder low-stringency conditions to obtain a full-length human ghrelincDNA. The human stomach cDNA library was prepared from human gastricpoly(A)⁺ RNA (Clontech Co., Ltd) by use of a cDNA synthesis kit(Pharmacia Co., Ltd). The full-length human ghrelin cDNA thus obtainedwas composed of 511 bases shown in SEQ ID NO: 7, coding a human ghrelinprecursor (prepro-ghrelin) consisting of 117 amino acids (FIG. 4 a).Homology at the amino acid sequence level between the rat- andhuman-derived ghrelin precursors was 82.9%, revealing that ghrelins arehighly conserved between species.

To known the distribution of ghrelin in tissues, poly(A)⁺ RNA isolatedfrom various rat tissues was analyzed (FIG. 4 b). By Northern blottinganalysis of the rat tissues, 0.62 kb ghrelin precursor mRNA wasrecognized in stomach. Two faint bands were also recognized inventricle, and these were 6.2 kb and 1.2 kb mRNAs which were larger thanthe mRNA in stomach, thus suggesting different mRNA splicing from thatin stomach. From the above result, it was found that a major expressionsite for ghrelin is stomach.

Example 6 Effect of Ghrelin on Secretion of Pituitary Hormones

Whether ghrelin has GH secretion-inducing activity or not was examinedin vitro and in vivo. First, the effect of ghrelin on primary culturedcells of anterior pituitary was examined for assay in vitro. Anteriorpituitaries were collected from 4-week-old male SD rats and dispersed bytreatment with collagenase, and the cells were collected, washed twicewith DMEM medium (Dulbecco's modified Eagle's medium) containing 10% FCS(fetal bovine serum) and an antibiotic, and suspended in DMEM medium toprepare the primary cultured cells of anterior pituitary at an initialstage. The 5×10⁴ cells were inoculated onto a 96-well cell culture platecoated with poly-D-lysine, and cultured for 3 to 4 days. The culturemedium was exchanged with DMEM medium containing 0.1 ml sample, andmaintained at 37° C. for 15 minutes. An aliquot of the culture mediumwas collected and measured by radioimmunoassay for the concentrations ofvarious pituitary hormones in the culture medium. Out of the pituitaryhormones, GH, FSH, LH, PRL and TSH were measured by using a kit producedby Biotrak/Amersham Co., Ltd, and ACTH was measured by using ahigh-sensitivity EIA kit produced by Peninsula Laboratories.

When ghrelin was added to the primary anterior pituitary, an increase inthe intracellular calcium ion concentration was recognized, whileunmodified synthetic ghrelin also showed an slightly increasedCa-releasing activity (FIG. 5 a). This result indicates that bothghrelin and unmodified ghrelin act directly on pituitary cells. Then,the GH secretion-inducing activity of ghrelin was examined using theprimary cultured of anterior pituitary cells at an initial stage, and byaddition of 10⁻⁶ M ghrelin, the concentration of GH in the culture wasincreased, but no increase in the concentrations of other pituitaryhormones (FSH, LH, PRL, TSH) was observed (FIG. 5 b).

The GH secretion-inducing activity of ghrelin was examined in vivo.After 10 μg of synthetic ghrelin was injected intravenously into a malerat (250 g), the blood was collected periodically for up to 60 minutesto measure the concentration of the pituitary hormones in plasma byradioimmunoassay. Out of the pituitary hormones, only GH was releasedinto blood and reached the maximum level in 5 to 10 minutes afterintravenous injection of ghrelin. From this result, it was found thatghrelin released from stomach to blood act on cells of anteriorpituitary and release GH into blood, and it was confirmed that theghrelin is an unidentified specific endogenous GH-secretion-inducingsubstance.

Example 7 Increase in Cardiac Output in Rat

The effect of bolus administration of ghrelin into a rat underanesthesia on the cardiovascular system was examined. Wistar male rats(Carerie) each weighing 220 to 250 g were divided at random into 4groups (as groups given 10, 1, 0.5, and 0.2 μg ghrelin respectively) inorder to examine the effect of acute administration of ghrelin on thecardiovascular system. Ghrelin was diluted with physiological saline andthen rapidly administered in a dose of 10, 1, 0.5, or 0.2 μg/rat/120 μlvia an injection tube (PE50) which had been inserted into the rightcommon jugular vein for measuring cardiac output.

As a dynamic indicator, systemic blood pressure and cardiac output weremeasured, and peripheral vascular resistance was calculated. The ratswere anesthetized with pentobarbital and fixed with dorsal position. Formeasurement of average blood pressure, a polyethylene canula (PE50)filled with heparin was inserted into the right femoral artery. Thecardiac output was measured using a thermal dilution-type cardiac outputmeter (CARDIOTHER M500R). An injection tube (PE50) filled withphysiological saline was inserted into the right common jugular vein inthe rat and retained in the right ventricle. A micro-catheter wasinserted into from the right common carotid artery in the rat andretained in the initiating part of the aorta. The infusion made use of100 μl physiological saline at room temperature (25° C.). By pushing aMEASURE switch of the thermal dilution-type cardiac output meter andsimultaneously injecting the infusion (100 μl physiological saline),measurement of cardiac output was initiated. Cardiac output was measured5 times to determine average cardiac output. Average blood pressure andcardiac output were determined in 1, 5, 15, and 30 minutes before andafter administration of ghrelin. Peripheral vascular resistance wasdetermined by dividing average blood pressure by cardiac output.

TABLE 1 Body Cardiac output (ml/min/kg) weight after administration of 1μg ghrelin (g) 0 min. 1 min. 5 min. 15 min. 30 min. Mean 230 347 382 367341 338 SEM 3.7 14.3 10.2 11.5 7.9 8.8 In the table, SEM is standarderror means.

TABLE 2 Body Cardiac output (ml/min/kg) weight after administration of10 μg ghrelin (g) 0 min. 1 min. 5 min. 15 min. 30 min. Mean 237 350 390392 370 344 SEM 1.0 8.5 7.4 15.8 14.7 13.8 In the Table, SEM is standarderror means.

In the group given 1 μg ghrelin (Table 1) and the group given 10 μgghrelin (Table 2), an increase in cardiac output was recognized in 1 to5 minutes after administration.

Example 8 Isolation of Ghrelin and Ghrelin-27 from Various Origins

From rat stomach extract, ghrelin was purified using Ca-releasingactivity as an indicator in the method described in Example 2. Theactive fraction (elution time of 59 minutes in FIG. 1 b) in secondaryCM-HPLC was purified to homogeneity by reverse phase HPLC onμBondasphere C-18 column (3.9×150 mm, produced by Waters Co., Ltd). Thisfraction was analyzed by electrospray ionization mass spectrometry(ESI-MS), indicating a peak of the molecular weight (3187.2±0.9) whichwas smaller by about 126 than that of natural ghrelin modified withoctanoic acid (C8) consisting of 28 amino acids. Determination of theamino acid sequence of this peptide by a peptide sequencer (ABI 494,manufactured by Applied Biosysytems Co., LTd) revealed that it is apeptide composed of the following 27 amino acid residues (SEQ ID NO:43): Gly Ser Xaa Phe Leu Ser Pro Glu His Gln Lys Ala Gln Arg Lys Glu SerLys Lys Pro Pro Ala Lys Leu Gln Pro Arg (Xaa is an unidentified aminoacid). That is, this peptide was composed of an amino acid sequencewherein in ghrelin consisting of 28 amino acids, glutamine 13 or 14 wasdeleted. Since the Ca-releasing activity of this peptide was similar tothat of ghrelin of 28 amino acids as shown in Example 9, this peptidewas designated as ghrelin-27. From human stomach extract, humanghrelin-27 was isolated in the same manner as for rat ghrelin, andconfirmed to consist of the amino acid sequence set forth in SEQ ID NO:11. Peak fractions with retention times of 64 to 65 minutes in secondaryCM-HPLC were purified and analyzed by electrospray ionization massspectrometry (ESI-MS), indicating a peak of the molecular weight(3341.4±0.9). Because this fatty acid-modified peptide was composed of28 amino acids, it was revealed to be a peptide wherein in ghrelin (28amino acids), 3rd serine was modified with decanoic acid (C10).

From the rat stomach cDNA library prepared in Example 5, a cDNA coding aprecursor of ghrelin-27 was cloned by plaque hybridization where thePCR-amplified DNA fragment prepared in Example 5 was used as a probe.The nucleotide sequence of the cDNA was determined and confirmed to codethe precursor of ghrelin-27. The resulting cDNA for the rat ghrelin-27precursor was composed of the nucleotide sequence set forth in SEQ IDNO: 14, coding the ghrelin-27 precursor having the amino acid sequence(116 amino acids) set forth in SEQ ID NO: 12. A cDNA for the humanghrelin-27 precursor was also cloned in the same manner as describedabove, and revealed to consist of the nucleotide sequence set forth inSEQ ID NO: 15, coding the human ghrelin-27 precursor having the aminoacid sequence (116 amino acids) set forth in SEQ ID NO: 13.

A cDNA coding a precursor of porcine-derived ghrelin or ghrelin-27 wascloned from a porcine cDNA library in the method described in Example 5by plaque hybridization where the PCR-amplified DNA fragment describedin Example 5 was used as a probe. The nucleotide sequence of theresulting cDNA clone was determined and confirmed to code a porcineghrelin precursor or a porcine ghrelin-27 precursor. The resulting cDNAfor the porcine ghrelin precursor was composed of the nucleotidesequence set forth in SEQ ID NO: 20, coding a ghrelin precursor havingthe amino acid sequence (118 amino acids) set forth in SEQ ID NO: 18.The cDNA for the porcine ghrelin-27 precursor was composed of thenucleotide sequence set forth in SEQ ID NO: 21, coding the ghrelin-27precursor having the amino acid sequence (117 amino acids) set forth inSEQ ID NO: 19. Accordingly, the porcine ghrelin (28 amino acids) and theporcine ghrelin-27 (27 amino acids) are composed of the amino acidsequences set forth in SEQ ID NOS: 16 and 17, respectively.

A cDNA coding a ghrelin precursor derived from eel, Xenopus Laevis orrainbow trout was cloned from various cDNA libraries in the methoddescribed in Example 5 by plaque hybridization where the PCR-amplifiedDNA fragment described in Example 5 was used as a probe. The nucleotidesequence of the resulting cDNA clone was determined and confirmed tocode the ghrelin precursor.

The resulting cDNA for the eel ghrelin precursor was composed of thenucleotide set forth in SEQ ID NO: 36, the cDNA for the frog ghrelinprecursor was composed of the nucleotide set forth in SEQ ID NO: 37, andthe cDNA for the rainbow trout ghrelin precursor was composed of thenucleotide set forth in SEQ ID NO: 38 or 39.

From rainbow trout, the cDNA coding the ghrelin-23 precursor set forthin SEQ ID NO: 38 and the cDNA coding the ghrelin-20 precursor set forthin SEQ ID NO: 39 were obtained.

From the nucleotide sequences of the above cDNAs, it was found that theeel ghrelin precursor has the amino acid sequence set forth in SEQ IDNO: 32, the frog ghrelin precursor has the amino acid sequence set forthin SEQ ID NO: 33, and the rainbow trout ghrelin precursor has the aminoacid sequence set forth in SEQ ID NO: 34 or 35.

From rainbow trout, the amino acid sequence of ghrelin-23 precursor setforth in SEQ ID NO: 34 and the amino acid sequence of ghrelin-20precursor in SEQ ID NO: 35 were found.

A cDNA for a bovine ghrelin precursor was cloned by the PCR method. Thatis, PCR was carried out where synthetic DNA having nucleotide sequencesdesigned on the basis of amino acid sequences conserved among the rat,human and porcine-derived ghrelins and ghrelins-27 were used as primerand a bovine stomach cDNA library as template. The DNA fragment thusamplified had the nucleotide sequence set forth in SEQ ID NO: 24, codinga part of bovine ghrelin-27 precursor set forth in SEQ ID NO: 23.Accordingly, the bovine ghrelin-27 has the amino acid sequence set forthin SEQ ID NO: 22. In the DNA fragment amplified by the above PCR usingthe bovine stomach cDNA library as template, there was no DNA coding aghrelin (28 amino acids) precursor.

The amino acids of the rat-, human- and porcine-derived ghrelins and therat-, human-, porcine- and bovine-derived ghrelins-27 were very similar,and in particular, the amino acid sequences of amino acids 1 to 10 inthe 7 ghrelins described above were completely identical with oneanother.

Example 9 Comparison of Activity Among Various Ghrelin Derivatives

Peptide fragments obtained by partially digested the rat- andhuman-derived ghrelins by various proteases, or chemically synthesizedpeptides, were examined for their Ca releasing activity in order todetermine a core amino acid sequence and the optimum chain length of amodifying fatty acid necessary for Ca-releasing activity. TheCa-releasing activity of ghrelin was expressed in terms of 50% effectiveconcentration (EC₅₀, nM) at which 50% of the maximum activity isachieved. Accordingly, lower EC₅₀ values are indicative of higheractivity.

TABLE 3 Comparison of the activity among various ghrelin derivatives SEQCa-releasing ID Amino Fatty acid activity Origin NO. acids modification(EC₅₀, nM) Remark human 3 1-28 Acyl (C:8) 2.6 natural ghrelin human 31-15 Acyl (C:8) 7.0 human 3 1-11 Acyl (C:8) 15 rat 2 1-28 Acyl (C:8) 2.9natural ghrelin rat 2 1-15 Acyl (C:8) 8.6 rat 2 1-11 Acyl (C:8) 15 rat 21-10 Acyl (C:8) 19 rat 2 1-9  Acyl (C:8) 38 rat 2 1-8  Acyl (C:8) 100rat 2 1-4  Acyl (C:8) 480 rat 2 16-28  Acyl (C:8) >10000 rat 2 (1-28) +Acyl (C:8) 2.8 ghrelin-27 (14-28) rat 2 1-28 Acyl (C:16) 3.1 rat 2 1-28Acyl (C:10) 2.6 rat 2 1-28 Acyl (C:6) 16 rat 2 1-28 Acyl (C:4) 280 rat 21-28 Acyl (C:2) 780

The Ca-releasing activity of ghrelin is present in the side of theamino-terminal. A peptide of from the amino-terminal to amino acid 4 hasa sufficient Ca-releasing activity, and a peptide of from theamino-terminal to amino acid 10 shows a strong Ca-releasing activitynear to that of natural ghrelin. When the chain length of modifyingfatty acid is C:2 (acetyl group), a sufficient activity is broughtabout, and when the chain length is C:8 (octanoyl group), the maximumCa-releasing activity is achieved, and even if the number of carbonatoms of fatty acid is further increased to C:10 (decanoyl group) or toC:16, the strong Ca-releasing activity does not change. That is, whenthe fatty acid for modifying 3rd serine from the amino-terminal contains8 or more carbon atoms, the strongest Ca-releasing activity is broughtabout.

Example 10 Synthesis of Various Ghrelin Derivatives (1) Synthesis ofPeptide Derivatives

Amino acid derivatives other than Fmoc-^(D)Ser (C₈H₁₇) and Fmoc-Ser(C₈H₁₇), and synthesis reagents, were purchased from Perkin Elmer,Novabiochem or Watanabe Kagaku Co., Ltd. Peptide chain extension wasperformed by mainly using Applied Biosystem 433A synthesizer produced byPerkin Elmer, and a protected peptide derivative-resin was constructedby the Boc or Fmoc method. The protected peptide resin obtained by theBoc method was deprotected with anhydrous hydrogen fluoride (HF) in thepresence of p-cresol thereby releasing the peptide, which was thenpurified. The protected peptide resin obtained by the Fmoc method wasdeprotected with trifluoroacetic acid (TFA) or dilute TFA containingvarious scavengers, and the released peptide was purified. Purificationwas performed in reversed phase HPLC on a C4 or C18 column. The purityof the purified product was confirmed by reverse phase HPLC, and itsstructure was confirmed by amino acid composition analysis and massspectrometry.

The peptide of the present invention is produced by a conventionalpeptide synthesis method. For example, it can be produced by a methoddescribed, in chapter 2 and 3 of “Biochemical Experimental Course 1,Protein Chemistry IV” (Tokyo KagakuDojin) or in “Development ofMedicines, a second series, Vol. 14, Peptide Synthesis (Hirokawa ShotenCo., Ltd). Accordingly, typical examples of the peptide of the presentinvention are shown below. Specifically, synthesis of acylated oralkylated peptides is exemplified below. Further, human-derived ghrelin(which may be abbreviated hereinafter to hGhrelin) or rat-derivedghrelin (which may be abbreviated hereinafter to rGhrelin) was reactedwith trypsin or chymotrypsin or both the enzymes successively to givethe following ghrelin fragments: 19. Ghrelin (16-28), 20. hGhrelin(1-15), 21. rGhrelin (1-15), 23. hGhrelin (1-11), 24. rGhrelin (1-11),25. Ghrelin (1-10), 26. Ghrelin (1-9), 27. Ghrelin (1-8), and 30.Ghrelin (1-4). Then, these fragments were isolated by analytical HPLCand measured for their activity. 41. [N-Acetyl]-Ghrelin (1-10) wasprepared in a usual manner by treating Ghrelin (1-10) withN-acetylsuccinimide. Compound No. 2 (rat ghrelin) made use of a naturalmaterial, and 10. [Ser³(Butyryl)]-rGhrelin, 11.[Ser³(Hexanoyl)]-rGhrelin, 12. [Ser³(Decanoyl)]-rGhrelin, 13.[Ser³(Lauroyl)]-rGhrelin, and 14. [Ser³(Palmitoyl)]-rGhrelin weresynthesized in the same manner as in synthesis of Compound 1 (hGhrelin)and then measured for their activity.

[Main Abbreviations]

HMP resin; 4-hydroxymethyl-phenoxymethyl resinFmoc amide resin; 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamido-ethyl resinPAM resin; phenylacetoamidomethyl resinHBTU; 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphateTBTU; 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborateHOBt; 1-hydroxybenzotriazoleDCC; dicyclohexylcarbodiimideDIPCI; diisopropylcarbodiimideTFA; trifluoroacetic acidDIPEA; diisopropylethylamineTIPS; triisopropylsilaneFmoc; fluorenylmethoxycarbonylBoc; t-butyloxycarbonylTrt; tritylBu^(t); t-butylPmc; 2,2,5,7,8-pentamethylchroman-6-sulfonylPrl; propionylPhPrl; phenylpropionylBzl; benzylBom; benzyloxymethylTos; toluenesulfonylCl-Z; 2-chloro-benzyloxycarbonylPis; 2-phenylisopropylMtt; 4-methyltrityl

DMF; N,N-dimethylformamide NMP; N-methylpyrrolidone

DMAP; 4-dimethylaminopyridine

HOSu; N-hydroxysucciniimide

Adod; 2-aminododecanoic acidAib; 2-aminoisobutylic acidApe; 5-aminopentanoic acidCha; cyclohexylalanineDap; 2,3-diaminopropionic acidNal; naphtylalanineNle; norleucine

[Protecting Amino Acids Used in Synthesis] Fmoc Method:

Boc-Gly, Fmoc-Gly, Fmoc-Ser (Bu^(t)), Fmoc-Ser (Trt), Fmoc-Glu(OBu^(t)), Fmoc-His (Boc), Fmoc-Gln (Trt), Fmoc-Arg (Pmc), Fmoc-Lys(Boc), Fmoc-Pro, Fmoc-Leu, Fmoc-Ala, Fmoc-Val, Fmoc-Phe, Fmoc-^(D)Phe,Fmoc-Ser (n-C₈H₁₇), Fmoc-^(D)Ser (n-C₈H₁₇), Fmoc-Cys (n-C₈H₁₇), Fmoc-Asp(OPis), Fmoc-Ser (Bzl), Fmoc-Cys (Trt), Fmoc-Dap (Octanoyl),Fmoc-2-^(L)Nal, Fmoc-2-^(D)Nal, Fmoc-Nle, Fmoc-Lys (Mtt), Fmoc-Aib-OH,Fmoc-Asp (O—C₇H₁₅)

Boc Method:

Boc-Gly, Boc-Ser (Bzl), Boc-Ser (Ac), Boc-Ser (Prl), Boc-Glu (OBzl),Boc-His (Bom), Boc-Gln, Boc-Arg (Tos), Boc-Lys (C1-Z), Boc-Pro, Boc-Leu,Boc-Ala, Boc-Val, Boc-Phe, Boc-Cys (n-C₈H₁₇), Boc-Ape Boc-Ser (n-C₈H₁₇)

[Units Used] (a) Analytical HPLC System Unit: Shimadzu LC-10A System

Column: YMC PROTEIN-RP (4.6 mmφ×150 mm)Column temperature: 40° C.Eluent: A linear gradient of from 0 to 50% acetonitrile for 20 minutesin 0.1% trifluoroacetic acidFlow rate: 1 mL/min.

Detection: UV (210 nm)

Injection volume: 10 to 100 μl

(b) Preparative HPLC System Unit: Waters 600 Multisolvent DeliverySystem

Columns: YMC-Pack-ODS-A (5 μm, 20 mm×250 mm)

-   -   YMC-Pack-PROTEIN-RP (5 μm, C4, 10 mm×250 mm)    -   YMC-Pack. PROTEIN-RP (5 μm, C4, 20 mm×250 mm)    -   YMC PROTEIN-RP (4.6 mmφ×150 mm)        Eluent: A suitable linear gradient of acetonitrile concentration        in 0.1% trifluoroacetic acid        Flow rate: 10 mL/min. (for the column of an inner diameter of 20        mm), 3 mL/min. (for the column of an inner diameter of 10 mm), 1        mL/min. (for the column of an inner diameter of 4.6 mm)

Detection: 210 nm, 260 nm

Injection: 10 to 2000 μl (2000 μl or more was injected via a pump)

(c) Mass Spectrometer Unit: Finigan MAT TSQ700

Ion source: ESIDetection ion mode: PositiveSpray voltage: 4.5 kVCapillary temperature: 250° C.Mobile phase: A mixture of 0.2% acetic acid and methanol (1:1)Flow rate: 0.2 mL/min.Scan range: m/z 300 to 1,500

(d) Analysis of Amino Acid Sequence

Unit: Applied Biosystem 477A, 492 model sequencer manufactured by PerkinElmer

(e) Analysis of Amino Acid Composition

Unit: L-8500 model amino acid analyzer manufactured by Hitachi, Co.,Ltd.Sample: Unless otherwise specified, the sample was hydrolyzed with 6 MHCl at 110° C. for 24 hours in a sealed tube.

(2) Example of Synthesis of a Derivative Having Acyl Serine or AcylThreonine (Fmoc Method, Carboxyl-Terminal Amide Derivatives)

Compound 1 hGhrelin: GSS(CO—C₇H₁₅) FLSPEHQRVQQRKESKKPPAKLQPR Fmoc-Arg(Pmc)-HMP-resin (403 mg, 0.25 mmol, ABI Co., Ltd) was treated with 20%piperazine for 20 minutes and subjected repeatedly to introduction ofFmoc-amino acid by HBTU/HOBt and elimination of Fmoc by piperazinesequentially to constructFmoc-Ser(Bu^(t))-Ser(Trt)-Phe-Leu-Ser(tBu)-Pro-Glu(OBu^(t))-His(Boc)-Gln(Trt)-Arg(Pmc)-Val-Gln(Trt)-Gln(Trt)-Arg(Pmc)-Lys(Boc)-Glu(OBu^(t))-Ser(Bu^(t))-Lys(Boc)-Lys(Boc)-Pro-Pro-Ala-Lys(Boc)-Leu-Gln(Trt)-Pro-Arg(Pmc)-resin.After Boc-Gly was finally introduced by DCC/HOBt, the resultingprotected peptide resin (1.3 g) was treated with 1% TFA-5%TIPS-methylene chloride solution (15 mL) for 30 minutes. The peptideresin was filtrated, washed several times with methylene chloride (30mL), and washed with 5% DIEA (10 mL) and then with methylene chloride(30 mL). The resulting de-Trt peptide resin (about 1.3 g) was swollenwith NMP (10 mL), and octanoic acid (144.2 mg, 1.0 mmol) and DIPCI(126.2 mg, 1.0 mmol) were added thereto in the presence of DMAP (61.1mg, 0.5 mmol) and allowed to react for 8 hours. The resin was recoveredby filtration and washed with NMP and then with methylene chloride,followed by drying under vacuum to give about 1.2 g protected peptideresin wherein the serine 3rd residue was octanoylated. To this productwas added a de-protecting reagent (10 mL) consisting of 88% TFA-5%phenol-2% TIPS-5% H₂O, and the mixture was stirred at room temperaturefor 2 hours. The resin was removed by filtration, and the filtrate wasconcentrated followed by adding ether to the resulting residues to formprecipitates. The precipitates were recovered by filtration and dried togive about 550 mg crude peptide. 200 mg of this product was dissolved in10 mL water and applied to YMC-Pack PROTEIN-RP (C4, 20 mm×250 mm) andeluted with a linear gradient (flow rate: 10 mL/min.) for 60 minutes offrom 0 to 54% acetonitrile in 0.1% trifluoroacetic acid. The desiredfractions were collected and lyophilized to give about 120 mg of thedesired product.

(3) Example of Synthesis of a Derivative Having Acyl Serine or AcylThreonine (Fmoc Method, Carboxyl-Terminal Amide Derivative) Compound 3Ghrelin (1-9)-NH₂; GSS(CO—C₂H₁₅)FLSPEH-NH₂

Fmoc-amide-resin (403 mg, 0.25 mmol, ABI Co., Ltd) was treated with 20%piperazine for 20 minutes and subjected repeatedly to introduction ofFmoc-amino acid by HBTU/HOBt and elimination of Fmoc by piperazinesequentially to constructFmoc-Ser(Bu^(t))-Ser(Trt)-Phe-Leu-Ser(Bu^(t))-Pro-Glu(OBu^(t))-His(Boc)-resin.After Boc-Gly was finally introduced by DCC/HOBt, the resultingprotected peptide resin (about 550 mg) was treated with 1% TFA-5%TIPS-methylene chloride solution (10 mL) for 30 minutes. The peptideresin was recovered by filtration, washed several times with methylenechloride (30 mL), and washed with 5% DIEA (10 mL) and then withmethylene chloride (30 mL). The resulting de-Trt peptide resin (about750 mg) was swollen with NMP (10 mL), and octanoic acid (144.2 mg, 1.0mmol) and DIPCI (126.2 mg, 1 mmol) were added thereto in the presence ofDMAP (61.1 mg, 0.5 mmol) and allowed to react for 4 hours. The resin wasrecovered by filtration and washed with NMP and then with methylenechloride, followed by drying under vacuum to give about 800 mg protectedpeptide resin wherein the 3rd serine residue was octanoylated. TFA (10mL) was added to this product and stirred at room temperature for 30minutes. The resin was removed by filtration, and the filtrate wasconcentrated followed by adding ether to the resulting residues to formprecipitates. The precipitates were recovered by filtration and dried togive about 250 mg crude peptide. About 200 mg of this product wasdissolved in 10 mL of 30% aqueous acetic acid and applied to YMC-PackPROTEIN-RP (C4, 20 mm×250 mm) and eluted with a linear gradient (flowrate: 10 mL/min.) for 60 minutes of from 0 to 54% acetonitrile in 0.1%trifluoroacetic acid. The desired fractions were collected andlyophilized to give about 150 mg of the desired product.

(4) Example of Synthesis of a Derivative Having Acyl Serine or AcylThreonine (Boc Method) Compound 9 (Ser³(Propionyl)-rGhrelin (1-28);GSS(CO—CH₂CH₃) FLSPEHQKAQQRKESKKPPAKLQPR

Protected rat ghrelin resin (4-28) was constructed from Boc-Arg(Tos)-Pam resin (0.75 g, 0.5 mmol) by Boc chemistry, and Boc-Ser(CO—CH₂CH₃)—OH, Boc-Ser (Bzl)-OH and Boc-Gly-OH were condensed with ahalf (1.4 g) of the resin. The resulting resin, 1.5 g, was treated witha mixture of HF and p-cresol (8.5 mL: 1.5 mL) at 0° C. for 1 hour, andthe HF was evaporated. Ether was added to the residues, whereby 671 mgcrude peptide was obtained. This sample was dissolved in 50% acetic acid(AcOH) and applied to a preparative column YMC-Pack-ODS-A (5 μm, 20mm×250 mm) and eluted at a rate of 10 mL/min. by a gradient of from 0 to95% acetonitrile concentration in 0.1% TFA solution for 75 minutes.Those fractions containing the desired product were lyophilized to give135.8 mg crude peptide. A part (0.5 mg) of this product was applied toYMC-A-302 column (C18, 4.6 mm×150 mm) and eluted at a flow rate of 1mL/min. by a gradient of from 15 to 19 concentration acetonitrile. Thispurification procedure was repeated and the desired fractions werecombined to give 0.41 mg of the desired product.

The following peptide derivatives having acyl serine or acyl threoninewere produced in the same manner as in production of Compound 3 or 9described above.

The results of the mass spectrometry and amino acid composition analysisof the peptide derivatives having acyl serine or acyl threonine aresummarized below.

Compound 1. hGhrelin

ESI-MS 3371.0 (theoretical: 3370.9), amino acid composition: Ser; 3.53(4), Glx; 5.91 (6), Gly; 1.02 (1), Ala; 1.00 (1), Val; 0.96 (1), Leu; 2,Phe; 1.06 (1), Lys; 3.90 (4), His; 0.97 (1), Arg; 2.87 (3), Pro; 3.87(4)

Compound 3. Ghrelin (1-9)-amide

ESI-MS [M+H]; 1085.7 (theoretical: 1085.2), amino acid composition: Ser;2.45 (3), Glx; 0.98 (1), Gly; 0.99 (1), Leu; 1, Phe; 0.99 (1), His; 1.08(1), Pro; 0.97 (1).

Compound 4. [Ser²(Octanoyl), Ser³]-Ghrelin (1-9)-amide

ESI-MS (M+H); 1085.8 (theoretical: 1085.2), amino acid composition: Ser;2.46 (3), Glx; 0.98 (1), Gly; 0.99 (1), Leu; 1, Phe; 1.01 (1), His; 1.09(1), Pro; 0.97 (1)

Compound 5. [Ser²(Octanoyl)]-Ghrelin (1-9)-amide

ESI-MS [M+H]; 1211.7 (theoretical: 1211.4), amino acid composition: Ser;2.48 (3), Glx; 1.00 (1), Gly; 1.01 (1), Leu; 1, Phe; 1.00 (1), His; 1.11(1), Pro; 0.98 (1)

Compound 8. [Ser³(Acetyl)]-rGhrelin

ESI-MS 3231.0 (theoretical: 3230.7), amino acid composition: Ser; 3.50(4), Glx; 5.90 (6), Gly; 0.98 (1), Ala; 2.00 (2), Leu; 2, Phe; 1.01 (1),Lys; 4.97 (5), His; 0.99 (1), Arg; 1.99 (2), Pro; 3.99 (4)

Compound 9. [Ser³(Propionyl)]-rGhrelin

ESI-MS 3245.0 (theoretical: 3242.8), amino acid composition: Ser; 3.42(4), Glx; 5.93 (6), Gly; 1.00 (1), Ala; 2.00 (2), Leu; 2, Phe; 1.10 (1),Lys; 4.97 (5), His; 0.99 (1), Arg; 1.99 (2), Pro; 3.83 (4)

Compound 15. [Ser³(3-Phenylpropionyl)]-hGhrelin

ESI-MS 3377.0 (theoretical: 3376.9), amino acid composition: Ser; 3.06(4), Glx; 5.92 (6), Gly; 0.93 (1), Ala; 0.98 (1), Val; 0.99 (1), Leu; 2,Phe; 1.13 (1), Lys; 4.03 (4), His; 1.08 (1), Arg; 3.00 (3), Pro; 3.76(4)

Compound 16. [Ser³(3-Octenoyl)]-hGhrelin

ESI-MS 3369.0 (theoretical: 3368.9), amino acid composition: Ser; 3.59(4), Glx; 5.91 (6), Gly; 1.00 (1), Ala; 1.02 (1), Val; 0.99 (1), Leu; 2,Phe; 1.15 (1), Lys; 3.97 (4), His; 0.98 (1), Arg; 2.93 (3), Pro; 3.88(4)

Compound 28. Ghrelin (1-8)-amide

ESI-MS [M+H] 948.5 (theoretical: 948.1), amino acid composition: Ser;2.45 (3), Glx; 0.97 (1), Gly; 0.99 (1), Leu; 1, Phe; 1.00 (1), Pro; 0.97(1)

Compound 29. Ghrelin (1-7)-amide

ESI-MS [M+H] 819.6 (theoretical: 819.0), amino acid composition: Ser;2.52 (3), Gly; 1.01 (1), Leu; 1, Phe; 1.02 (1), Pro; 1.09 (1)

Compound 30. Ghrelin (1-6)-amide

ESI-MS [M+H]; 722.4 (theoretical: 721.8), amino acid composition: Ser;2.47 (3), Gly; 0.99 (1), Leu; 1, Phe; 1.00 (1)

Compound 31. Ghrelin (1-5)

ESI-MS [M+H] 636.5 (theoretical: 635.8), amino acid composition: Ser;1.78 (2), Gly; 0.99 (1), Leu; 1, Phe; 1.02 (1)

Compound 32. Ghrelin (1-5)-amide

ESI-MS [M+H] 635.4 (theoretical: 634.8), amino acid composition: Ser;1.67 (2), Gly; 1.01 (1), Leu; 1, Phe; 1.01 (1)

Compound 33-2. Ghrelin (1-4)-amide

ESI-MS [M+H] 522.2 (theoretical: 521.6), amino acid composition: Ser;1.65 (2), Gly; 0.99 (1), Phe; 1

Compound 34. Ghrelin (1-3)-amide

ESI-MS [M+H] 375.2 (theoretical: 374.4), amino acid composition: Ser;1.66 (2), Gly; 1 Compound 35. [Lys⁸]-Ghrelin (1-8)-amide

ESI-MS [M+H] 947.9 (theoretical: 947.1), amino acid composition: Ser;2.70 (3), Gly; 1.00 (1), Leu; 1, Phe; 1.00 (1), Lys; 0.99 (1), Pro; 1.00(1)

Compound 36. [Arg⁸]-Ghrelin (1-8)-amide

ESI-MS [M+H] 975.8 (theoretical: 975.2), amino acid composition: Ser;2.70 (3), Gly; 1.00 (1), Leu; 1, Phe; 1.01 (1), Arg; 0.99 (1), Pro; 1.00(1)

Compound 37. [Lys⁶]-Ghrelin (1-6)-amide

ESI-MS [M+H] 763.6 (theoretical: 762.9), amino acid composition: Ser;1.80 (2), Gly; 1.00 (1), Leu; 1, Phe; 1.01 (1), Lys; 1.00 (1)

Compound 38. [Lys⁵]-Ghrelin (1-5)-amide

ESI-MS [M+H] 650.5 (theoretical: 649.8), amino acid composition: Ser;1.79 (2), Gly; 0.99 (1), Phe; 1, Lys; 0.99 (1)

Compound 39. [^(D)Phe⁴, Lys⁵]-Ghrelin (1-5)-amide

ESI-MS [M+H] 650.5 (theoretical: 649.8), amino acid composition: Ser;1.79 (2), Gly; 0.99 (1), Phe; 1, Lys; 0.99 (1)

Compound 40. [N-Aminopentanoyl]-Ghrelin (3-7)-amide

ESI-MS [M+H] 774.7 (theoretical: 774.0), amino acid composition: Ser;1.80 (2), Leu; 1, Phe; 1.01 (1), Pro; 1.00 (1)

Compound 43. [N-Glycyl]-Ghrelin (3-7)-amide

ESI-MS [M+H]; 732.7 (theoretical: 731.9), amino acid composition: Ser;1.80 (2), Gly; 1.00 (1), Leu; 1, Phe; 1.01 (1), Pro; 1.00 (1)

Compound 44. [Leu²]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 845.7 (theoretical: 845.1), amino acid composition: Ser;1.80 (2), Gly; 1.01 (1), Leu; 2, Phe; 1.02 (1), Pro; 0.99 (1)

Compound 45. [His²]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 869.7 (theoretical: 869.0), amino acid composition afterhydrolysis with propionic acid.hydrochloric acid (50/50) at 150° C. for2 hours: Ser; 1.02 (2), Gly; 1.00 (1), Leu; 1, Phe; 1.00 (1), His; 0.95(1), Pro; 0.99 (1)

Compound 46. [Lys²]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 860.7 (theoretical: 860.1), amino acid composition afterhydrolysis with propionic acid.hydrochloric acid (50/50) at 150° C. for2 hours: Ser; 1.04 (2), Gly; 1.00 (1), Leu; 1, Phe; 1.00 (1), Lys; 1.00(1), Pro; 1.00 (1)

Compound 47. [Gly²]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 789.5 (theoretical: 788.9), amino acid composition afterhydrolysis with propionic acid.hydrochloric acid (50/50) at 150° C. for2 hours: Ser; 1.14 (2), Gly; 2.01 (2), Leu; 1, Phe; 1.00 (1), Pro; 1.00(1)

Compound 59. [Thr²(Octanoyl)]-hGhrelin

ESI-MS M; 3384.0 (theoretical: 3384.9), amino acid composition: Ala;1.02 (1) Arg; 2.99 (3), Glx; 5.91 (6), Gly; 1.02 (1), His; 1.00 (1),Leu; 2 (2), Lys; 4.05 (4), Phe; 1.00 (1), Pro; 4.06 (4), Ser; 2.66 (3),Thr; 0.94 (1), Val; 0.96 (1)

Compound 60. [Leu², Thr³(Octanoyl)]-hGhrelin

ESI-MS M; 3410.0 (theoretical: 3411.0), amino acid composition: Ala;1.01 (1), Arg; 2.95 (3), Glx; 5.92 (6), Gly; 1.01 (1), His; 1.01 (1),Leu; 3 (3), Lys; 4.02 (4), Phe; 1.01 (1), Pro; 4.00 (4), Ser; 1.81 (2),Thr; 0.96 (1), Val; 0.97 (1)

Compound 69. [Ser²(4-Methylpentanoyl)]-hGhrelin

ESI-MS M; 3343.0 (theoretical: 3342.9), amino acid composition: Ala;1.00 (1), Arg; 2.97 (3), Glx; 5.86 (6), Gly; 1.02 (1), His; 1.01 (1),Leu; 2, Lys; 4.00 (4), Phe; 1.01 (1), Pro; 3.99 (4), Ser; 3.54 (4), Val;0.98 (1)

Compound 75. [Lys⁷]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 850.5 (theoretical: 850.0), amino acid composition: Ser;2.67 (3), Gly; 1.00 (1), Leu; 1, Phe; 1.00 (1), Lys; 1.00 (1)

(5) Example of Synthesis of an Amino-Terminal Acylated Derivative

Compound 6. [N-Octanoyl, Ser³]-Ghrelin (1-9)-amide;C₇H₁₅CO-GSSFLSPEH-NH₂

Fmoc-amide resin (403 mg, 0.25 mmol, ABI Co., Ltd) was treated with 20%piperazine for 20 minutes and subjected repeatedly to introduction ofFmoc-amino acid by HBTU/HOBt and elimination of Fmoc by piperazinesequentially to constructFmoc-Gly-Ser(Bu^(t))-Ser(Bu^(t))-Phe-Leu-Ser(tBu)-Pro-Glu(OBu^(t))-His(Boc)-resin.After treatment with piperazine, the resulting resin (550 mg) was washedwith NMP, and DIPCI (126.2 mg, 1 mmol) and octanoic acid (144.2 mg, 1.0mmol) were added thereto in the presence of HOBt (135.1 mg, 1 mmol) andallowed to react for 4 hours. The resin was recovered by filtration,washed with NMP and then with methylene chloride, and dried under vacuumto give about 600 mg protected peptide resin wherein the amino group inamino-terminal Gly was octanoylated. This product was deprotected withTFA (10 ml) (treatment for 30 minutes), to give 200 mg crude peptide.The whole of the sample was applied to YMC-Pack PROTEIN-RP (5 μm, C4, 20mm×250 mm) and eluted with a linear gradient (flow rate: 10 mL/min) for60 minutes of from 0 to 54% acetonitrile in 0.1% trifluoroacetic acid.About 180 mg of the desired product was obtained.

Measured values: ESI-MS [M+H]; 1085.6 (theoretical: 1085.2), amino acidcomposition: Ser; 2.47 (3), Glx; 0.98 (1), Gly; 1.00 (1), Leu; 1, Phe;1.02 (1), His; 1.09 (1), Pro; 0.96 (1)

(6) Example of Synthesis of a Derivative Containing Serine Having anAlkyl Side-Chain

Compound 50. [Ser³(Octyl)]-Ghrelin (1-7)-amide; GSS(C₈H₁₇) FLSP-NH₂

Fmoc-Ser (C₈H₁₇)

Under cooling on ice, sodium hydride (3.19 g, 133 mmol) was added to asolution of Boc-Ser (12.3 g, 53.9 mmol) in DMF (300 ml) and stirred atroom temperature for 1.5 hours. Octane iodide (11.0 ml, 60.9 mmol) wasadded thereto and stirred at room temperature for 16 hours. After water(40 ml) was added dropwise to the reaction solution under cooling onice, the solvent was evaporated under vacuum. The resulting residueswere purified by applying them to silica gel column chromatography (gel;Art9385, Merck Co., Ltd, eluent; dichloromethane:methanol:aceticacid=120:10:1), to give 6.88 g Boc-Ser (C₈H₁₇) (yield, 36.2%) as paleyellow oily matter. Trifluoroacetic acid (120 ml) was added to thisproduct, Boc-Ser (C₈H₁₇) (6.88 g, 21.7 mmol), under cooling on ice andstirred for 0.5 hour at room temperature. After the trifluoroacetic acidwas evaporated, the resulting residues were dissolved in diethyl ether(120 ml), and 4N HCl-dioxane (22 ml) was added thereto and stirred for 1hour under cooling on ice. The precipitated crystals were recovered byfiltration to give 5.23 g H-Ser (C₈H₁₇).HCl (yield, 96.3%) as colorlesscrystals. After triethylamine (1.40 ml, 10 mmol) was added to asuspension (50 ml) of this product H-Ser (C₈H₁₇).HCl (2.54 g, 10.0 mmol)in 10 sodium hydrogen carbonate, a solution of Fmoc-Osu (5.00 g, 14.8mmol) in 1,2-dimethoxyethane (20 ml) was added dropwise thereto over theperiod of 10 minutes and stirred at room temperature for 16 hours. Theinsolubles were filtered off, then dichloromethane was added to thefiltrate, and the organic phase was separated and washed with 13% NaClsolution. The organic phase was dried over anhydrous sodium sulfate, andthe solvent was evaporated. The resulting residues were purified byapplying them to silica gel column chromatography (gel; BW-300, FujiSilicia Co., Ltd, eluent; dichloromethane:methanol=93:7), to give 2.75 gFmoc-Ser (C₈H₁₇) (yield: 62.6%) as colorless crystals. Rf=0.45 (CHCl₃:MeOH=9:1, Silica gel 60F₂₅₄, MERCK Co., Ltd). Fmoc-^(D)Ser(C₈H₁₇):Rf=0.45 (CHCl₃: MeOH=9:1, Silica gel 60F₂₅₄, MERCK Co., Ltd).

Fmoc-amide resin (400 mg, 0.25 mmol, ABI Co., Ltd) was treated with 20%piperazine for 20 minutes and subjected repeatedly to introduction ofFmoc-amino acid by HBTU/HOBt and elimination of Fmoc by piperazinesequentially to constructFmoc-Ser(Bu^(t))-Ser(C₈H₁₇)-Phe-Leu-Ser(Bu^(t))-Pro-resin. After Boc-Glywas finally introduced by DCC/HOBt, a part (250 mg) of the resultingprotected peptide resin was treated with TFA (10 mL) for 30 minutes. Theresin was removed by filtration, and the filtrate was concentratedfollowed by adding ether to the resulting residues to give about 120 mgcrude peptide as precipitates. This product was dissolved in 5% AcOH (10mL) and applied to YMC-Pack-ODS-A (5 μm, 20 mm×250 mm) and eluted with alinear gradient (flow rate: 10 mL/min.) for 60 minutes of from 0 to 60%acetonitrile in 0.1% trifluoroacetic acid. The desired fractions werecollected and lyophilized to give about 40 mg of the desired product.

Compound 84. [N-Aminopentanoyl, Ser³(Octyl)]-Ghrelin (3-5)-benzyl amide;

H-Ape-Ser(C₈H₁₇)-Phe-Leu-NH—CH₂-Ph

Oxime resin (230 mg/0.25 mmol, Novabiochem Co., Ltd) was placed in areaction vessel equipped with a glass filter, and Boc-Leu-OH.H₂O (190mg, 0.75 mmol) previously dissolved in methylene chloride (DCM) anddried over MgSO₄, DCC (160 mg, 0.75 mmol), and 5 ml DCM were addedthereto and shaken overnight. The reaction product was washed severaltimes with a suitable amount of DCM, DCM/EtOH (1:1) and DCM in thisorder. After introduction of Leu, <1> 10 ml of 25% TFA/DCM was addedthereto and shaken for 30 minutes, and the resin was washed severaltimes with DCM, isopropyl alcohol (iPrOH), DCM and DMF in this order,and <2> a solution prepared by dissolving 0.75 mmol (3 equivalents) ofBoc-amino acid, 0.75 mmol (3 equivalents) of TBTU and 0.75 mmol (3equivalents) of HOBt and then adding 1.25 mmol (5 equivalents) of DIPEAthereto in 5 mL DMF in an Erlenmeyer flask was introduced into thereaction vessel and shaken for 1 hour; this operation was repeatedlycarried out to condense amino acids sequentially. Finally,Boc-NH—(CH₂)₄—CO-Ser(C₈H₁₇)-Phe-Leu-Oxime resin, 370 mg, was obtained.The resin was suspended in about 5 mL DMF, and benzylamine hydrochloride(180 mg, 1.25 mmol), triethylamine (173 μL, 1.25 mmol) and acetic acid(72 μL, 1.25 mmol) were added thereto, and the mixture was stirred.After 24 hours, the resin was filtered off, and the filtrate wasevaporated, and the resulting Boc-protected peptide was precipitated in10 mL of 1N HCl. This product was washed with water and dried, and 5 mLTFA was added thereto and reacted for 30 minutes thereby eliminate theBoc. The TFA was evaporated, and the product was precipitated with ether(Et₂O), whereby the desired product (N-Aminopentanoyl,Ser³(Octyl)-Ghrelin (3-5)-benzylamide, 110 mg, was obtained. Compounds82, 83 and 85 were synthesized in the same manner.

The following peptide derivatives having alkyl serine, except forCompounds 82 to 85, were produced in the same manner as in production ofCompound 50 described above.

The results of the mass spectrometry and amino acid composition analysisof the peptide derivatives having alkyl serine are summarized below.

Compound 17. [Ser³(Octyl)]-hGhrelin

ESI-MS; 3357.0 (theoretical: 3356.9), amino acid composition: Ser; 2.92(3+1), Glx; 5.94 (6), Gly; 1.00 (1), Ala; 0.98 (1), Val; 0.99 (1), Leu;2, Phe; 1.13 (1), Lys; 4.04 (4), His; 1.09 (1), Arg; 3.01 (3), Pro; 3.89(4)

Compound 50. [Ser³(Octyl)]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 805.5 (theoretical: 805.0), amino acid composition afterhydrolysis with propionic acid.hydrochloric acid (50/50) at 150° C. for2 hours: Ser; 0.86 (2+1), Gly; 1.01 (1), Leu; 1, Phe; 1.06 (1), Pro;0.95 (1)

Compound 51. [Ser³(Octyl), ^(D)Phe⁴]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 805.4 (theoretical: 805.0), amino acid composition afterhydrolysis with propionic acid.hydrochloric acid (50/50) at 150° C. for2 hours: Ser; 0.97 (2+1), Gly; 1.00 (1), Leu; 1, Phe; 1.05 (1), Pro;1.16 (1)

Compound 52. [^(D)Ser³ (Octyl)]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 805.4 (theoretical: 805.0), amino acid composition afterhydrolysis with propionic acid.hydrochloric acid (50/50) at 150° C. for2 hours: Ser; 1.51 (2+1), Gly; 1.00 (1), Leu; 1, Phe; 1.00 (1), Pro;1.00 (1)

Compound 53. [^(D)Ser³(Octyl), ^(D)Phe⁴]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 805.5 (theoretical: 805.0), amino acid composition afterhydrolysis with propionic acid.hydrochloric acid (50/50) at 150° C. for2 hours: Ser; 1.51 (2+1), Gly; 1.00 (1), Leu; 1, Phe; 1.00 (1), Pro;1.01 (1)

Compound 67. [Ser³(Bzl)]-hGhrelin

ESI-MS M; 3335.0 (theoretical: 3334.8), amino acid composition: Ala;1.00 (1), Arg; 2.96 (3), Glx; 5.92 (6), Gly; 1.00 (1), His; 1.01 (1),Leu; 2 (2), Lys; 4.00 (4), Phe; 1.02 (1), Pro; 4.08 (4), Ser; 3.58 (4),Val; 0.98 (1)

Compound 76. [N-Aminopentanoyl, Ser³(Octyl), Lys⁵]-Ghrelin (3-5)-amide

ESI-MS [M+H]; 591.5 (theoretical: 590.8), amino acid composition: Ser;0.45 (1), Phe; 1, Lys; 1.00 (1)

Compound 77. [N-Aminopentanoyl, ^(D)Ser³(Octyl), ^(D)Phe⁴, Lys⁵]-Ghrelin(3-5)-amide

ESI-MS [M+H]; 591.5 (theoretical: 590.8), amino acid composition: Ser;0.45 (1), Phe; 1, Lys; 1.01 (1)

Compound 78. [Aib¹, His², Ser³(Octyl), Lys⁵]-Ghrelin (1-5)-amide

ESI-MS [M+H]; 714.6 (theoretical: 713.9), amino acid composition: Ser;0.45 (1), Phe; 1, His; 1.01 (1), Lys; 1.00 (1)

Compound 79. [Aib¹, His², ^(D)Ser³(Octyl), ^(D)Phe⁴, Lys⁵]-Ghrelin(1-5)-amide

ESI-MS [M+H]; 714.5 (theoretical: 713.9), amino acid composition: Ser;0.44 (1), Phe; 1, His; 1.00 (1), Lys; 1.01 (1)

Compound 81. [N-Aminopentanoyl, Ser³(Octyl)]-Ghrelin (3-5)-amide

ESI-MS [M+H]; 576.5 (theoretical: 575.8), amino acid composition: Ser;0.49 (1), Leu; 1, Phe; 0.99 (1)

Compound 82. [N-Aminopentanoyl, Ser³(Octyl)]-Ghrelin (3-5)-methylamide

ESI-MS [M+H]; 590.6 (theoretical: 589.8), amino acid composition: Ser;0.49 (1), Leu; 1, Phe; 0.99 (1)

Compound 83. [N-Aminopentanoyl, Ser³(Octyl)]-Ghrelin (3-5)-ethylamide

ESI-MS [M+H]; 604.3 (theoretical: 603.8), amino acid composition: Ser;0.50 (1), Leu; 1, Phe; 0.99 (1)

Compound 84. [N-Aminopentanoyl, Ser³(Octyl)]-Ghrelin (3-5)-benzylamide

ESI-MS [M+H]; 666.5 (theoretical: 665.9), amino acid composition: Ser;0.46 (1), Leu; 1, Phe; 0.98 (1)

Compound 85. [N-Aminopentanoyl, Ser³(Octyl)]-Ghrelin(3-5)-aminoethylamide

ESI-MS [M+H]; 619.6 (theoretical: 618.9), amino acid composition: Ser;0.47 (1), Leu; 1, Phe; 0.99 (1)

(7) Example of Synthesis of a Derivative Containing Cysteine Having anAlkyl Side-Chain Compound 48. [Cys³(Octyl)]-Ghrelin (1-7)-NH₂;GSC(C₈H₁₇)FLSP-NH₂

Fmoc-amide-resin (403 mg, 0.25 mmol, ABI Co., Ltd) was treated with 20%piperazine for 20 minutes and subjected repeatedly to introduction ofFmoc-amino acid by HBTU/HOBt and elimination of Fmoc by piperazinesequentially to constructFmoc-Ser(Bu^(t))-Cys(C₈H₁₇)-Phe-Leu-Ser(Bu^(t))-Pro resin. After Boc-Glywas finally introduced by DCC/HOBt, the resulting protected peptideresin (550 mg) was treated with TFA (10 mL) for 30 minutes. The resinwas removed by filtration, and the filtrate was concentrated followed byadding ether to the resulting residues to give about 120 mg crudepeptide as precipitates. This product was dissolved in 10 mL of 5%acetic acid and applied to YMC-Pack-ODS-A (5 μm, 20 mm×250 mm) andeluted with a linear gradient (flow rate: 10 mL/min.) for 60 minutes offrom 0 to 60% acetonitrile in 0.1% trifluoroacetic acid. The desiredfractions were collected and lyophilized to give about 44 mg of thedesired product.

Compound 68. [Cys³(Trt)]-hGhrelin; GSC(C-Ph₃)FLSPEHQRVQQRKESKKPPAKLQPR

Fmoc-Arg (Pmc)-resin (403 mg, 0.25 mmol, ABI Co., Ltd) was treated with20% piperazine for 20 minutes and subjected repeatedly to introductionof Fmoc-amino acid by HBTU/HOBt and elimination of Fmoc by piperazinesequentially to constructFmoc-Ser(Bu^(t))-Cys(Trt)-Phe-Leu-Ser(tBu)-Pro-Glu(OBu^(t))-His(Boc)-Gln(Trt)-Arg(Pmc)-Val-Gln(Trt)-Gln(Trt)-Arg(Pmc)-Lys(Boc)-Glu(OBu^(t))-Ser(Bu^(t))-Lys(Boc)-Lys(Boc)-Pro-Pro-Ala-Lys(Boc)-Leu-Gln(Trt)-Pro-Arg(Pmc)-HMPresin. After Boc-Gly was finally introduced by DCC/HOBt, the resultingprotected peptide resin (1.4 g) was recovered. TFA (15 mL) was added toa part (400 mg) of the resulting resin and stirred at room temperaturefor 1 hour. The resin was removed by filtration, and the filtrate wasconcentrated followed by adding ether to the resulting residues to formprecipitates. About 90 mg of the precipitates were dissolved in 40 mLwater, then applied to YMC-Pack PROTEIN-RP (C4, 20 mm×250 mm) and elutedwith a linear gradient (flow rate: 10 mL/min.) for 60 minutes of from 0to 54% acetonitrile in 0.1% trifluoroacetic acid. The desired fractionswere collected and lyophilized to give about 60 mg of the desiredproduct.

The following peptide derivatives having alkyl cysteine were produced inthe same manner as in production of Compound 48 or 68 described above.

The results of the mass spectrometry and amino acid composition analysisof the peptide derivatives having alkyl cysteine are summarized below.

Compound 18. [Cys³(Octyl)]-rGhrelin

ESI-MS; 3317.0 (theoretical: 3316.9), amino acid composition: Ser; 2.69(3), Glx; 5.90 (6), Gly; 1.00 (1), Ala; 1.99 (2), Leu; 2, Phe; 1.02 (1),Lys; 4.97 (5), His; 0.99 (1), Arg; 1.98 (2), Pro; 3.87 (4)

Compound 48. [Cys³ (Octyl)]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 821.7 (theoretical: 821.1), amino acid composition afterhydrolysis with propionic acid.hydrochloric acid (50/50) at 150° C. for2 hours: Ser; 0.60 (2), Gly; 1.08 (1), Leu; 1, Phe; 1.06 (1), Pro; 0.96(1)

Compound 49. [Cys³(Octyl), ^(D)Phe⁴]-Ghrelin (1-7)-amide

ESI-MS [M+H]; 821.6 (theoretical: 821.1), amino acid composition afterhydrolysis with propionic acid.hydrochloric acid (50/50) at 150° C. for2 hours: Ser; 0.58 (2), Gly; 1.02 (1), Leu; 1, Phe; 1.06 (1), Pro; 0.97(1)

Compound 68. [Cys³(Trt)]-hGhrelin

ESI-MS 3503.0 (theoretical: 3503.1), amino acid composition: Ser; 2.42(3), Glx; 5.77 (6), Gly; 1.00 (1), Ala; 1.01 (1), Val; 0.94 (1), Leu; 2,Phe; 0.99 (1), Lys; 3.94 (4), His; 0.99 (1), Arg; 2.92 (3), Pro; 3.81(4)

(8) Example of Synthesis of a Peptide Derivative Containing N-MethylAmino Acids

Compound 86. [N-Aminopentanoyl, Ser³(Octyl), MePhe⁴, MeLeu⁵]-Ghrelin(3-5)-amide; NH₂— (CH₂)₄—CO-Ser (C₈H₁₇)-MePhe-MeLeu-NH₂

Fmoc-amide resin (0.40 g, 0.25 mmol) was placed in a reaction vesselequipped with a glass filter, and 15 mL of 20% piperidine in NMP wasadded thereto and shaken for 20 minutes, thus removing the Fmoc group.Thereafter, 15 mL NMP, 1.0 mmol (4 equivalents) of Fmoc-MeLeu-OH, 1.0mmol (4 equivalents) of TBTU, 1.0 mmol (4 equivalents) of HOBt and 1.0mmol (4 equivalents) of DIPEA were added thereto and shaken for 1 hourto condense the Fmoc-MeLeu. Thereafter, the peptide chain was extendedby repeatedly carrying out removal of Fmoc group by 20% piperidine andcondensation of Fmoc-amino acid (3 equivalents) bybromo-tris-pyrrolidino-phosphonium hexafluorophosphate (3 equivalents)in the presence of 2.25 mmol (9 equivalents) of DIPEA. The conclusion ofthe condensation reaction was confirmed by deprotecting a small amountof the resin with TFA and examining it by HPLC and mass spectrometry(MS). After Boc-NH—(CH₂)₄—CO-Ser(O—C₈H₁₇)-MePhe-MeLeu-resin wasobtained, this resin was treated with TFA for 30 minutes, whereby theresin was cleaved to de-protect the peptide. After the TFA wasevaporated, the peptide was washed with ether (Et₂O) to give 120 mgNH₂—(CH₂)₄—CO-Ser(C₈H₁₇)-MePhe-MeLeu-NH₂. This product was applied toYMC-Pack ODS-A (C18, 20 mm×250 mm) and eluted with a linear gradient(flow rate: 10 mL/min.) for 60 minutes of 0 to 54% acetonitrile in 0.1%trifluoroacetic acid. The desired fractions were collected andlyophilized to give 70 mg of the desired product. After this derivativewas hydrolyzed with propionic acid.HCl (50/50) at 150° C. for 2 hours,the amount of the peptide was quantified using the ratio of the peakarea of aminopentanoic acid detected in the amino acid analyzer to thatof 10 nmol aminopentanoic acid as a standard.

ESI-MS [M+H]; 604.5 (theoretical: 603.8), detected amino acids afterhydrolysis with propionic acid.HCl (50/50) at 150° C. for 2 hours: Ser,Ape.

(9) Synthesis of a Mixed-Disulfide Derivative

Compound 57. [Cys³(S-Heptyl)]-hGhrelin; GSC(S—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR

A de-protecting reagent (15 mL) consisting of 88% TFA-5% phenol-2%TIPS-5% H₂O was added to a protected peptide-HMP resin (1 g) obtained bysynthesis in the same manner as in production of Compound 68, and thenstirred at room temperature for 2 hours. The resin was removed byfiltration, and the filtrate was concentrated followed by adding etherto the resulting residues, whereby about 550 mg crude [Cys³]-hGhrelinpowder was obtained. This product was applied to YMC-Pack ODS-A (C18, 20mm×250 mm) and eluted with a linear gradient (flow rate: 10 mL/min.) for60 minutes of from 0 to 54% acetonitrile in 0.1% trifluoroacetic acid.The desired fractions were collected and lyophilized to give 300 mg[Cys³]-hGhrelin (1-28). 40 mg (11.4 μmol) of this product was dissolvedin water (20 mL), and 1 mL solution of 4,4′-dithiodipyridine (7.5 mg,34.2 μmol) in acetonitrile was added thereto and left for 1 hour. Afterthe conclusion of the reaction was confirmed, the reaction solution waswashed several times with chloroform to remove an excess of the4,4′-dithiodipyridine and pyridone derivative. The aqueous layer (10 mL)containing [thiopyridyl Cys³]-hGhrelin (1-28) was adjusted to pH 7.4with aq. 5% NH₃, and a solution of 1-heptane [sic.] thiol (4.5 mg, 34.2μmol) in 2 mL acetonitrile was added thereto. After 1 hour, the reactionsolution was applied to YMC-Pack ODS-A (C18, 20 mm×250 mm) and elutedwith a linear gradient (flow rate: 10 mL/min) for 60 minutes of from 0to 54% acetonitrile in 0.1% trifluoroacetic acid. The desired fractionswere collected and lyophilized to give 15 mg of the desired product.

Compound 57. [Cys³(S-Heptyl)]-hGhrelin

ESI-MS 3391.0 (theoretical: 3391.0), amino acid composition: Ser; 2.76(3), Glx; 5.81 (6), Gly; 0.99 (1), Ala; 1.01 (1), Val; 0.95 (1), Leu; 2,Phe; 0.99 (1), Lys; 3.95 (4), His; 0.99 (1), Arg; 2.93 (3), Pro; 3.84(4)

(10) Examples of Synthesis of a Derivative Having an Amide in a SideChain at the 3rd Position and an Ester in the Reverse Direction

Compound 55. [Asp³(NH-Heptyl)]-hGhrelin;GSD(NH—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR

Fmoc-Arg(Pmc)-HMP-resin (403 mg, 0.25 mmol, ABI Co., Ltd) was treatedwith 20% piperazine for 20 minutes and subjected repeatedly tointroduction of Fmoc-amino acid by HBTU/HOBt and elimination of Fmoc bypiperazine sequentially to constructFmoc-Ser(Bu^(t))-Asp(OPis)-Phe-Leu-Ser(tBu)-Pro-Glu(OBu^(t))-His(Boc)-Gln(Trt)-Arg(Pmc)-Val-Gln(Trt)-Gln(Trt)-Arg(Pmc)-Lys(Boc)-Glu(OBu^(t))-Ser(Bu^(t))-Lys(Boc)-Lys(Boc)-Pro-Pro-Ala-Lys(Boc)-Leu-Gln(Trt)-Pro-Arg(Pmc)-HMPresin. After Boc-Gly was finally introduced by DCC/HOBt, the resultingprotected peptide resin (1.3 g) was treated with 4% TFA-methylenechloride solution (15 mL) for 15 minutes. The peptide resin wasrecovered by filtration and washed several times with methylene chloride(30 mL), washed with 4% DIEA (10 mL) and then with methylene chloride(30 mL).

The resulting de-Pis peptide resin (about 1.3 g) was swollen with NMP(10 mL), and water-soluble carbodiimide hydrochloride (191.7 mg, 1.0mmol), HOBt (135.2 mg, 1.0 mmol) and n-heptylamine (115.2 mg, 1.0 mmol)were added thereto and allowed to react for 8 hours.

The resin was recovered by filtration, washed with NMP and methylenechloride, and dried under vacuum to give about 1.2 g protected peptideresin where the Asp 3 residue was heptylamidated. A deprotecting reagent(10 mL) consisting of 88% TFA-5% phenol-2% TIPS-5% H₂O was added theretoand stirred at room temperature for 2 hours. The resin was removed byfiltration, and the filtrate was concentrated followed by adding etherto the resulting residues to form precipitates. The precipitates wererecovered by filtration and dried to give about 550 mg crude peptide.

200 mg of this product was dissolved in 10 mL water and applied toYMC-Pack PROTEIN-RP (C4, 20 mm×250 mm) and eluted with a linear gradient(flow rate: 10 mL/min.) for 60 minutes of from 0 to 54% acetonitrile in0.1% trifluoroacetic acid. The desired fractions were collected andlyophilized to give about 120 mg of the desired product.

Compound 61. [Lys³(Octanoyl)]-hGhrelin; GSK(CO—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR

Fmoc-Arg (Pmc)-HMP-resin (403 mg, 0.25 mmol, a product of ABI Co., Ltd)was treated with 20% piperazine for 20 minutes and subjected repeatedlyto introduction of Fmoc-amino acid by HBTU/HOBt and elimination of Fmocby piperazine sequentially to constructBoc-Gly-Ser(tBu)-Lys(Mtt)-Phe-Leu-Ser(tBu)-Pro-Glu(OBu^(t))-His(Boc)-Gln(Trt)-Arg(Pmc)-Val-Gln(Trt)-Gln(Trt)-Arg(Pmc)-Lys(Boc)-Glu(OBu^(t))-Ser(Bu^(t))-Lys(Boc)-Lys(Boc)-Pro-Pro-Ala-Lys(Boc)-Leu-Gln(Trt)-Pro-Arg(Pmc)-HMPresin. About 300 mg of the resulting protected peptide resin was treatedwith 1% TFA-5% TIPS-methylene chloride solution (15 mL) for 60 minutes.

The peptide resin was recovered by filtration and washed several timeswith methylene chloride (30 mL), washed with 10% DIEA (10 mL) and thenwith methylene chloride (30 mL). The resulting de-Mtt peptide resin(about 300 mg) was swollen with NMP (2 mL), and octanoic acid (40 μl,0.25 mmol) and DCC (52 mg, 0.25 mmol) were added thereto in the presenceof HOBt (34 mg, 0.25 mmol) and allowed to react overnight.

The resin was recovered by filtration, washed with NMP and then withmethylene chloride, and dried under vacuum to give about 300 mgprotected peptide resin where the lysine 3rd residue was octanoylated. Adeprotecting reagent (5 mL) consisting of 88% TFA-5% phenol-2% TIPS-5%H₂O was added thereto and stirred at room temperature for 2 hours. Theresin was removed by filtration, and the filtrate was concentratedfollowed by adding ether to the resulting residues to form precipitates.The precipitates were separated by filtration and dried to give about234 mg crude peptide.

This product was dissolved in 6 mL acetic acid and applied to YMC-PackODS-A (5 μm, 20 mm×250 mm) and eluted with a linear gradient (flow rate:10 mL/min.) for 60 minutes of from 0 to 60% acetonitrile in 0.1%trifluoroacetic acid. The desired fractions were collected andlyophilized to give about 100 mg powder. This product was dissolved in 2mL of 50% acetic acid and applied to YMC-Pack PROTEIN-RP (5 μm, C4, 20mm×250 mm) and eluted with a linear gradient (flow rate: 10 mL/min.) for60 minutes of from 0 to 60% acetonitrile in 0.1% trifluoroacetic acid.The desired fractions were collected and lyophilized to give about 52 mgpowder.

The following compounds were produced in the same manner as inpreparation of Compound 55 or 61 described above.

The results of the mass spectrometry and amino acid composition analysisof the peptide derivatives synthesized by the conventional Fmoc methodare summarized below.

Compound 54. [Asp³(O-Heptyl)]-hGhrelin (1-28)

ESI-MS 3371.0 (theoretical: 3370.9), amino acid composition: Asx; 0.99(1), Ser; 2.70 (3), Glx; 5.87 (6), Gly; 1.01 (1), Ala; 1.01 (1), Val;0.94 (1), Leu; 2, Phe; 1.00 (1), Lys; 4.02 (4), His; 1.00 (1), Arg; 2.98(3), Pro; 3.84 (4)

Compound 55. [Asp³(NH-Heptyl)]-hGhrelin (1-28)

ESI-MS 3370.0 (theoretical: 3369.9), amino acid composition: Asx; 0.88(1), Ser; 2.95 (3), Glx; 5.97 (6), Gly; 1.21 (1), Ala; 1.03 (1), Val;0.98 (1), Leu; 2, Phe; 1.00 (1), Lys; 3.94 (4), His; 0.92 (1), Arg; 2.91(3), Pro; 3.99 (4)

Compound 56. [Dap³(Octanoyl)]-hGhrelin

ESI-MS M; 3370.0 (theoretical: 3369.9), amino acid composition: Ala;1.02 (1), Arg; 2.94 (3), Glx; 5.94 (6), Gly; 1.00 (1), His; 0.91 (1),Leu; 2 (2), Lys; 3.93 (4), Phe; 0.99 (1), Pro; 4.01 (4), Ser; 2.88 (3),Val; 0.98 (1), Dap; N.D.

Compound 58. [Adod³]-hGhrelin (1-28)

ESI-MS M; 3355.0 (theoretical: 3355.0), amino acid composition: Ala;1.01 (1), Arg; 2.91 (3), Glx; 5.95 (6), Gly; 1.01 (1), His; 0.91 (1),Leu; 2 (2), Lys; 3.94 (4), Phe; 0.99 (1), Pro; 4.02 (4), Ser; 2.88 (3),Val; 0.96 (1)

Compound 61. [Lys³(Octanoyl)]-hGhrelin

ESI-MS M; 3412.0 (theoretical: 3412.0), amino acid composition: Ala;1.05 (1), Arg; 3.05 (3), Glx; 6.02 (6), Gly; 1.00 (1), His; 1.00 (1),Leu; 2 (2), Lys; 5.11 (5), Phe; 0.97 (1), Pro; 4.20 (4), Ser; 2.68 (3),Val; 1.00 (1)

Compound 62. [Trp³]-hGhrelin

ESI-MS M; 3343.0 (theoretical: 3343.9), amino acid composition: Ala;1.00 (1), Arg; 3.03 (3), Glx; 5.94 (6), Gly; 1.01 (1), His; 1.01 (1),Leu; 2 (2), Lys; 4.00 (4), Phe; 0.99 (1), Pro; 3.96 (4), Ser; 2.60 (3),Trp; N.D., Val; 0.98 (1)

Compound 63. [Phe³]-hGhrelin

ESI-MS M; 3305.0 (theoretical: 3304.8), amino acid composition: Ala;0.99 (1), Arg; 2.96 (3), Glx; 5.86 (6), Gly; 1.00 (1), His; 1.00 (1),Leu; 2 (2), Lys; 3.98 (4), Phe; 2.01 (2), Pro; 3.99 (4), Ser; 2.67 (3),Val; 0.98 (1)

Compound 64. [Cha³]-hGhrelin

ESI-MS M; 3411.0 (theoretical: 3410.9), amino acid composition: Ala;1.02 (1), Arg; 3.01 (3), Glx; 5.92 (6), Gly; 1.01 (1), His+Cha; 2.01(1+1), Leu; 2 (2), Lys; 4.02 (4), Phe; 1.01 (1), Pro; 4.03 (4), Ser;2.72 (3), Val; 0.97 (1)

Compound 65. [2-^(L)Nal³]-hGhrelin

ESI-MS M; 3354.0 (theoretical: 3354.9), amino acid composition: Ala;1.00 (1), Arg; 2.95 (3), Glx; 5.87 (6), Gly; 1.02 (1), His; 1.01 (1),Leu; 2 (2), Lys; 3.98 (4), Phe; 1.01 (1), Pro; 3.94 (4), Ser; 2.73 (3),Val; 0.97 (1), Nal; N.D. (1)

Compound 66. [2-^(D)Nal³]-hGhrelin

ESI-MS M; 3355.0 (theoretical: 3354.9), amino acid composition: Ala;1.02 (1), Arg; 2.95 (3), Glx; 5.96 (6), Gly; 1.00 (1), His; 0.92 (1),Leu; 2 (2), Lys; 3.94 (4), Phe; 0.99 (1), Pro; 4.02 (4), Ser; 2.91 (3),Val; 0.98 (1), Nal; N.D. (2)

Compound 70. [Leu^(3])-hGhrelin

ESI-MS M; 3270.0 (theoretical: 3270.8), amino acid composition: Ala;0.99 (1), Arg; 2.95 (3), Glx; 5.88 (6), Gly; 1.01 (1), His; 1.00 (1),Leu; 3 (3), Lys; 3.96 (4), Phe; 1.00 (1), Pro; 3.89 (4), Ser; 2.65 (3),Val; 0.97 (1)

Compound 71. [Ile³]-hGhrelin

ESI-MS M; 3270.0 (theoretical: 3270.8), amino acid composition: Ala;0.98 (1), Arg; 2.96 (3), Glx; 5.87 (6), Gly; 0.99 (1), His; 1.01 (1),Ile; 0.98 (1), Leu; 2 (2), Lys; 3.97 (4), Phe; 1.00 (1), Pro; 3.97 (4),Ser; 2.65 (3), Val; 0.98 (1)

Compound 72. [Lys³(Octanoyl)]-hGhrelin

ESI-MS M; 3286.0 (theoretical: 3285.8), amino acid composition: Ala;1.02 (1), Arg; 2.94 (3), Glx; 5.95 (6), Gly; 0.99 (1), His; 0.92 (1),Leu; 2 (2), Lys; 4.92 (5), Phe; 0.99 (1), Pro; 4.02 (4), Ser; 2.91 (4),Val; 0.99 (1)

Compound 73. [Nle³]-hGhrelin

ESI-MS M; 3270.0 (theoretical: 3270.8), amino acid composition: Ala;1.01 (1), Arg; 2.98 (3), Glx; 5.92 (6), Gly; 1.02 (1), His; 1.01 (1),Leu; 2 (2), Lys; 4.01°(4), Phe; 1.01 (1), Pro; 4.01 (4), Ser; 2.71 (3),Val; 0.98 (1), Nle; N.D. (1)

Compound 74. [Val³]-hGhrelin

ESI-MS M; 3256.0 (theoretical: 3256.8), amino acid composition: Ala;0.98 (1), Arg; 2.96 (3), Glx; 5.84 (6), Gly; 1.00 (1), His; 1.01 (1),Leu; 2 (2), Lys; 3.97 (4), Phe; 0.99 (1), Pro; 3.94 (4), Ser; 2.64 (3),Val; 1.97 (2)

Compound 80. [Aib¹, His², ^(D)Nal³, ^(D)Phe⁴, Lys⁵]-Ghrelin (1-5)-amide;Ipamorelin

ESI-MS [M+H]; 712.5 (theoretical: 711.9), amino acid composition: Phe;1, His; 1.00 (1), Lys; 1.00 (1)

Example 11 Comparison of Activity Among Ghrelin Derivative Peptide-TypeCompounds

The Ca-increasing activities of the ghrelin derivative peptide-typecompounds synthesized in Example 10 and the natural ghrelin peptide weremeasured in the same manner as in Example 1.

(1) Modification of a Side Chain of Serine 3 A. Position of OctanoylGroup

The significant structural feature of ghrelin lies in the octanoyl groupon the hydroxyl group of serine 3. First, whether or not it isadvantageous for exhibiting the activity

Example 11 Comparison of Activity Among Ghrelin Derivative Peptide-TypeCompounds

The Ca-increasing activities of the ghrelin derivative peptide-typecompounds synthesized in Example 10 and the natural ghrelin peptide weremeasured in the same manner as in Example 1.

(1) Modification of a Side Chain of 3rd Serine A. Position of anOctanoyl Group

The significant structural feature of ghrelin lies in the octanoyl groupon the hydroxyl group of 3rd serine. First, whether or not it isadvantageous for exhibiting the activity that the position of serine tobe octanoylated is the 3rd position was examined. In this examination,the intracellular Ca-releasing activity in CHO cells expressing rat GSHreceptor was used as the indicator.

On the basis of ghrelin (1-9) amide (a short-chain ghrelin derivative)whose EC₅₀ value was kept at 5.4 nM, [serine² (octanoyl),serine³]-ghrelin (1-9) amide, [serine² (octanoyl)]-ghrelin (1-9) amide,and (N^(α)-octanoyl, serine³)-ghrelin (1-9) amide were synthesized, andtheir intracellular Ca-releasing activity was examined.

The results are summarized in Table 4.

TABLE 4 Ghrelin derivative activity 1 Ca- releasing Compound activityStructure EC₅₀ (nM) 1. human Ghrelin 1.3GSS(CO—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR 2. rat Ghrelin 1.5GSS(CO—C₇H₁₅)FLSPEHQKAQQRKESKKPPAKLQPR 3. Ghrelin (1-9)-amide 5.4H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-His-NH₂ 4. [Ser²(Octanoyl),Ser³]-Ghrelin (1-9)-amide 1,100H-Gly-Ser(CO—C₇H₁₅)-Ser-Phe-Leu-Ser-Pro-Glu-His-NH₂ 5.[Ser²(Octanoyl)]-Ghrelin (1-9)-amide 1,400H-Gly-Ser(CO—C₇H₁₅)-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro- Glu-His-NH₂ 6.[N-Octanoyl, Ser³]-Ghrelin (1-9)-amide >10,000C₇H₁₅CO-Gly-Leu-Ser-Phe-Leu-Ser-Pro-Glu-His-NH₂

The activity was reduced to about 1/200 by transferring an octanoylgroup from 3rd serine to 2nd serine in human ghrelin (EC₅₀=1,100 nM).

The derivative having octanoyl groups at both the 2nd and 3rd positionsalso showed a reduced activity (EC₅₀=1,400 nM).

Further, the activity was relatively weakened by N-octanoylation at onlythe amino-terminal amino group (EC₅₀>10,000 nM).

From these results, it was revealed that the position of the amino acidmodified with an octanoyl group is particularly preferably the 3rdposition in the ghrelin molecule.

B. Chain Length of a Fatty Acid

The intracellular Ca-releasing activity of the des-octanoyl derivativederived from rat ghrelin by eliminating the side-chain octanoyl group of3rd serine was 3,500 nM as compared with the activity (2.6 nM) of theoctanoylated ghrelin, and thus it is evident that the side-chainoctanoyl group of 3rd serine plays a very important role in expressingthe activity.

Accordingly, the relationship between the activity and the number ofcarbon atoms in the side-chain acyl group of serine in rat ghrelin wasexamined using various saturated fatty acids. That is, the intracellularCa-releasing activities of the ghrelin derivatives wherein the hydroxylgroup of 3rd serine was acylated with an acetyl group (CH₃CO—),propionyl group (CH₃CH₂CO—) butyryl group (CH₃(CH₂)₂CO—), hexanoyl group(CH₃(CH₂)₄CO—) decanoyl group (CH₃(CH₂)₈CO—), lauroyl group(CH₃(CH₂)₁₀CO—), and palmitoyl group (CH₃(CH₂)₁₄CO—) were determined.

The results are summarized in Table 5.

TABLE 5 Ghrelin derivative activity 2 Ca- releasing Compound activityStructure EC₅₀ (nM) 7. [Ser³]-rat Ghrelin 3,500GSSFLSPEHQKAQQRKESKKPPAKLQPR 8. [Ser3 (Acetyl)]-rGhrelin 780GSS(CO—CH₃)FLSPEHQKAQQRKESKKPPAKLQPR 9. [Ser³ (Propionyl)]-rGhrelin n.t. GSS(CO—C₂H₅)FLSPEHQKAQQRKESKICPPAKLQPR 10. [Ser³ (Butyryl)]-rGhrelin280 GSS(CO—C₃H₇)FLSPEHQKAQQRKESKKPPAKLQPR 11. [Ser³ (Hexanoyl)]-rGhrelin16 GSS(CO—C₅H₁₁) FLSPEHQKAQQRKESKKPPAKLQPR 12. [Ser³ (Decanoyl)-rGhrelin1.7 GSS(CO—C₉H₁₉)FLSPEHQKAQQRKESKKPPAKLQPR 13. [Ser³ (Lauroyl)]-rGhrelin2.4 GSS(CO—C₁₁H₂₃)FLSPEHQKAQQRKESKKPPAKLQPR 14.[Ser³(Palmitoyl)]-rGhrelin 6.5 GSS(CO—C₁₅H₃₁)FLSPEHQKAQQRKESKKPPAKLQPRIn the Table, ″n. t.″ indicates that the sample was not tested.

The influence of the chain length of fatty acid on the activity was madeincreasingly significant with an EC₅₀ value of 780 nM for the ghrelinderivatives having acetyl group (C2) and an EC₅₀ value of 280 nM for theghrelin derivatives having butanoyl group (C4), and the ghrelinderivatives having the hexanoyl group (C7) brought about a furtherincrease in the Ca-releasing activity (EO₅₀ value, 16 nM), and theghrelin the octanoyl group permitted the Ca-releasing activity to reacha peak (EO₅₀ value, 1.5 nM). Even the ghrelin derivatives having thedecanoyl group (C10) maintained a similar Ca-releasing activity (EO₅₀value, 1.7 nM) to that of ghrelin, and further the EC₅₀ value was 2.4 nMfor the ghrelin derivatives having lauroyl group (C12) and 6.5 nM forthe ghrelin derivatives having palmitoyl group (C16), thus indicatingthat the Ca-releasing activity was maintained even if the chain lengthof fatty acid was increased.

C. Substitution of Various Acyl Groups

Human ghrelin derivatives were prepared by binding 3-phenyl propionicacid (HO—CO—CH₂CH₂Ph) as a typical example of aromatic fatty acid,3-octenoic acid (OH₃(CH₂)₃CH═CH—CH₂COH) as a typical example ofunsaturated fatty acid or 4-methyl pentanoic acid ((CH₃)₂CH—CH₂CH₂CO₂H)as a typical example of branched fatty acid, in place of saturated fattyacid, via an ester linkage to the hydroxyl group of 3rd serine, andtheir activity was examined.

D. Conversion into Alkyl Groups

By converting the chemically instable ester linkage into a chemicallystable ether or thioether linkage or the like, chemically stable ghrelinderivatives can be formed. However, it goes without saying thatmaintenance of the activity is a preposition for this conversion.

Hence, an ether derivative of human ghrelin wherein 3rd serine wasoctylated (C₈H₁₇) and a thioether derivative of rat ghrelin wherein 3rdserine was replaced by cysteine and octylated were examined for theiractivity.

Further, a derivative of human ghrelin wherein 3rd serine was benzylated(—CH₂Ph) and a derivative of human ghrelin wherein 3rd serine wasreplaced by cysteine and tritylated (—C(Ph)₃) were prepared.

The results are summarized in Table 6. The Ca-releasing activities ofthe derivative of human ghrelin wherein 3rd serine was benzylated(—CH₂Ph) and the derivative of human ghrelin wherein 3rd serine wasreplaced by cysteine and tritylated (—C(Ph)₃) are shown as those ofCompounds 67 and 68 respectively in Table 13. The Ca-releasing activityof the derivative of human ghrelin wherein 4-methyl pentanoic acid((CH₃)₂CH—CH₂CH₂CO₂H) was bound via an ester linkage to the hydroxylgroup of 3rd serine is also shown as that of Compound 69 in Table 13.

TABLE 6 Ghrelin derivative activity 3 Ca- releasing Compound activityStructure EC₅₀ (nM) 15. (Ser³(3-Phenylpropionyl)]-hGhrelin 1.4GSS(CO—CH₂CH₂Ph)FLSPEHQRVQQRKESKKPPAKLQPR 16.(Ser³(3-Octenoyl)]-hGhrelin 1.7 GSS(CO—CH₂CH═CH(CH₂)₃CH₃₎)FLSPEHQRVQQRKESKKPPAKLQPR 17. (Ser³(Octyl)]-hGhrelin 1.2GSS(C₈H₁₇)FLSPEHQRVQQRKESKKPPAKLQPR 18. [Cys³(Octyl)]-rGhrelin 5.4GSC(C₈H₁₇)FLSPEHQKAQQRKESKKPPAKLQPR

Introduction of 3-octenoyl group as an example of unsaturated fatty acidinto the side-chain of 3rd serine brought about a similar Ca-releasingactivity (EC₅₀=1.7 nM) to the activity of the ghrelin derivatives havingan octanoyl group.

Interestingly, even if a phenyl propionyl group was introduced, theCa-releasing activity was maintained to be high (EC₅₀=1.4 nM), and evenif a 4-methylpentanoyl group (C6) as an example of branched fatty acidwas introduced, the EC₅₀ value was 4.4 nM, indicating that theCa-releasing activity was maintained (Compound 69 in Table 13), thusrevealing that it is not always necessary that the side-chain acyl groupin 3rd serine is a linear-chain alkanoyl group.

Further, the EC₅₀ values of the ether and thioether derivativesexpectable to be chemically stable, wherein 3rd serine or 3rd cysteinewere octylated, were maintained to be 1.2 nM and 5.4 nM respectively,thus revealing that it is not always necessary that the side chain ofamino acid residue at 3rd position is an acyl group.

Further, the EC₅₀ values of ghrelins wherein amino acid residue 3rdposition was replaced by Ser (Bzl) [that is, the derivative of humanghrelin wherein 3rd serine was benzylated (—CH₂Ph)] or by Cys (Trt)[that is, the derivative of human ghrelin wherein 3rd serine wasreplaced by cysteine and tritylated (—C(Ph)₃)] were 7.6 nM and 20 nM,respectively, thus indicating that the Ca-releasing activity wasmaintained (Compounds 67 and 68 in Table 13).

(2) Determination of the Active Region

The intracellular Ca-releasing activity of ghrelin (16-28) containingthe original carboxyl-terminal region was relatively low (EC₅₀>10,000nM), while the EC₅₀ values of human ghrelin (1-15) and rat ghrelin(1-15) both containing the original amino-terminal region were 7.0 nMand 8.6 nM respectively, thus indicating that the intracellularCa-releasing activity was maintained, and it was thereby revealed thatthe active site of ghrelin is present in the amino-terminal region(Table 7).

TABLE 7 Ghrelin derivative activity 4 Ca- releasing Compound activityStructure EC₅₀ (nM) 19. Ghrelin (16-28) >10,000H-Lys-Glu-Ser-Lys-Lys-Pro-pro-Ala-lysLeu-Gln-Pro-Arg- OH 20. hGhrelin(1-15) 7.0 H-Gly-Ser-Ser (CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-OH 21. rGhrelin (1-15) 8.6H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-His-Gln-Lys-Ala-Gln-Gln-Arg-OH 22. (des Gln¹⁴)-rGhrerin 1.5GSS(CO—C₇H₁₅)FLSPEHQKAQ_RKESKKPPAKLQPR

Further, because the activities of human and rat ghrelins (1-15) werealmost the same, the amino acid resides 11th and 12th position(arginyl-valyl- in human, and -lysyl-aranyl- in rat) are not limited tothese amino acids.

The results of the correlation between structure and activity, obtainedusing human or rat ghrelin, can be applied to rat and human ghrelinsrespectively.

Further, [des-glutamine¹⁴]-rat ghrelin prepared by removing 14thglutamine from the ghrelin exhibited a Ca-releasing activity (EC₅₀=1.5nM) similar to that of rat ghrelin, indicating that the amino acid inthe middle of the ghrelin molecule may be deleted.

(3) Peptide Chain Length and Introduction of Basic Group into theCarboxyl-Terminal

On the basis of ghrelin (1-15) found to have a relatively strongactivity, a derivative was prepared by suitably deletingcarboxyl-terminal amino acid residues from the ghrelin(1-15), and theiractivity was evaluated.

The activities of the short-chain derivatives having carboxylic acid atthe carboxyl-terminal and the short-chain derivatives amidated at thecarboxyl-terminal are shown in Table 8.

TABLE 8 Ghrelin derivative activity 5 Ca- releasing Compound activityStructure EC₅₀ (nM) 23. hGhrelin (1-11) 15H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-His-Gln- Arg-OH 24. rGhrelin(1-11) 15 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-His-Gln- Lys-OH25. Ghrelin (1-10) 19H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-His-Gln- OH 26. Ghrelin(1-9) 38 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-His-OH 27. Ghrelin(1-8) 100 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-OH 28. Ghrelin(1-8)-amide 13 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-NH₂ 29.Ghrelin (1-7)-amide 2.6 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-NH₂ 30.Ghrelin (1-6)-amide 4.8 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-NH₂ 31.Ghrelin (1-5) 68 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-OH 32. Ghrelin(1-5)-amide 6.2 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-NH₂ 33-1. Ghrelin (1-4)480 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-OH 33-2. Ghrelin (1-4)-amide 160H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-NH₂ 34. Ghrelin (1-3)-amide >10,000H-Gly-Ser-Ser(CO—C₇H₁₅)—NH₂

The Ca-releasing activity of ghrelin (1-3) amide was relatively low(EC₅₀>10,000 nM). The EC₅₀ of ghrelin (1-4) added phenylalanine to theghrelin(1-3) was 480 nM and the EC₅₀ of the carboxyl-terminal amidederivative thereof was 160 nM, thus revealing that they have asignificant Ca-releasing activity.

Further, the activity of ghrelin (1-5) amide added leucine amide toghrelin(1-4) was about 26 times (EC₅₀=6.2 nM) as high as that ofghrelin(1-4) amide, thus exhibiting a Ca-releasing activity at the samelevel as that of natural ghrelin.

The highest Ca-releasing activity was found in ghrelin (1-7) amide, andits EC₅₀ value was almost equivalent to that of natural ghrelin.

From the above result, the structural factor essential for expressingthe ghrelin activity could be attributed to the sequence of 4thamino-terminal residues, but because its affinity for ghrelin receptoror signal transduction is drastically improved by adding a residue suchas leucine at the 5th position, a residue such as leucine is preferablyadded at the 5th position.

As is evident from the above result, the Ca-releasing activity tended tobe increased by amidation of carboxyl-terminal carboxylic acid.

For example, the Ca-releasing activity (EC₅₀=5.4 nM) of ghrelin (1-9)after amidation was about 7 times as high as the activity (EC₅₀=38 nM)before amidation, and the Ca-releasing activity (EC₅₀=160 nM) of ghrelin(1-4) after amidation was about 3 times as high as the activity(EC₅₀=480 nM) before amidation. Further, the Ca-releasing activity(EC₅₀=13 nM) of ghrelin (1-8)amide produced from ghrelin (1-9)amide byremoving basic histidine reside 9 was lower than the activity (EC₅₀=5.4nM) of ghrelin (1-9)amide, while the Ca-releasing activity (EC₅₀=2.6 nM)of ghrelin (1-7) amide produced by removing glutamic acid 8 as acidicamino acid was higher than the activity (EC₅₀=13 n14) before removal.

One effect of amidation is to neutralize the negative charge ofcarboxylic acid, and the above result indicates that the basicity ofcarboxyl-terminal amino acid in the short-chain derivative contributessignificantly to the increase in activity.

On the basis of this result, derivatives endowed with basicity at thecarboxyl-terminal, which are similar to ghrelin (1-7) amide showing highactivity, were prepared and their activity was examined.

The results are shown in Table 9.

TABLE 9 Ghrelin derivative activity 6 Ca- releasing Compound activityStructure EC₅₀ (nM) 35. [Lys⁸]-Ghrelin (1-8)-amide H-Gly-Ser-Ser 1.1(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Lys-NH₂ 36. [Are⁸]-Ghrelin (1-8)-amideH-Gly-Ser-Ser 1.1 (CO—C₇H₁₅)-Phe-Leu-Ser-Pro-Arg-NH₂ 37. [Lys⁶]-Ghrelin(1-6)-amide 12 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Lys-NH₂ 38.[Lys⁵]-Ghrelin (1-5)-amide 10 H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Lys-NH₂ 39.[^(D)Phe⁴, Lys⁵]-Ghrelin (1-5)-amide 1,700 H-Gly-Ser-Ser(CO—C₇H₁₅)- _(D)Phe-Lys-NH₂

The Ca-releasing activity (EC₅₀=12 nM) of [lysine⁶]-ghrelin (1-6) amidehaving lysine added at the carboxyl-terminal of ghrelin (1-5) wasslightly lower than the activity (EC₅₀=4.8 nM) of ghrelin (1-5), whilethe Ca-releasing activity (EC₅₀=10 nM) of ghrelin (1-4) having lysineadded at the carboxyl-terminal was about 50 times as high as theactivity (EC₅₀=480 nM) before addition. Further, the Ca-releasingactivity (EC₅₀=1.1 nM), respectively, of the amide derivative havingarginine or lysine added at the carboxyl-terminal of ghrelin (1-7) wasvery stronger than the activity (EC₅₀=2.6 nM) of ghrelin (1-7) amide.

It was revealed that in almost all the cases, the activity is increasedby masking of acidity at the carboxyl-terminal and introduction of abasic group.

(4) Amino-Terminal Glycine and 2nd Serine Residue

On the basis of ghrelin (1-7) amide (EC₅₀=2.6 nM) [Compound 29 in Table8] or ghrelin (1-9) amide (EC₅₀=5.4 nM) [Compound 3 in Table 4] found tohave activity, the influence of amino-terminal glycine and 2nd serine onthe activity was examined.

The results are summarized in Table 10.

TABLE 10 Ghrelin derivative activity 7 Ca- releasing Compound activityStructure EC₅₀ (nM) 40. [N-Aminopentanoyl]-Ghrelin (3-7)-amide 3.4NH₂—(CH₂) ₄—CO-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-NH₂ 41. [N-Acetyl]-Ghrelin(1-10) >10,000 CH₃CO-Gly-Ser-Ser(CO-C₇H₁₅)-Phe-Leu-Ser-Pro-Glu-His-Gln-OH 42. [N-Tyr]-rGhrelin 120 YGSS(CO-C₇H₁₅)FLSPEHQKAQQRKESKKPPAKLQPR 43.[N-Glycyl]-Ghrelin (3-7)-amide 380H-Gly-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-NH₂ 44. [Leu²]-Ghrelin (1-7)-amide42 H-Gly-Leu-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-NH₂ 45. [His²]-Ghrelin(1-7)-amide 35 H-Gly-His-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-NH₂ 46.[Lys²]-Ghrelin (1-7)-amide 24 H-Gly-Lys-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-NH₂ 47. [Gly²]-Ghrelin (1-7)-amide 78H-Gly-Gly-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Pro-NH₂

The activity of N^(α)-acetyl-ghrelin (1-10) wherein the amino-terminalamino group in the ghrelin(1-10) was blocked was relatively low(EC₅₀>10,000 nM). As described above, the activity of (N^(α)-ocatanoyl,serine³)-ghrelin (1-9) amide (Compound 6 in Table 1) was also relativelylow (EC₅₀>10,000 nM), and thus the amino-terminal amino group ispreferably not blocked in order to express the Ca-releasing activity.

On the other hand, the Ca-releasing activity ofN^(α)-aminopentanoyl-ghrelin (3-7) amide wherein amino-terminal glycineand 2nd serine were replaced by 5-amino-n-pentanoic acid(NH₂—(CH₂)₄—CO—) having a length of 2nd residues was almost maintained(EC₅₀=3.4 nM), the Ca-releasing activity of [N^(α)-glycyl]-ghrelin (3-7)amide from which 2nd serine had been deleted was lower (EC₅₀=380 nM),and the Ca-increasing activity of [N-tyrosyl]-rat ghrelin having atyrosine residue added at the amino-terminal in the rat ghrelin waslower (EC₅₀=120 nM) so that it is preferable for stronger activity thatthe amino-terminal amino group is positioned such that amino-terminalamino acid(s) having a length of 2 residues are present from octanoyl3rd serine residue to the amino-terminal.

Further, the EC₅₀ values of the derivatives of ghrelin (1-7) amidewherein 2nd serine had been replaced by leucine, glycine, histidine andlysine were 42 nM, 78 nM, 35 nM and 24 nM respectively, indicating aslightly lower Ca-releasing activity than that of ghrelin (1-7) amide.

Since this result indicates that the 2nd serine residue(—NH—CH(CH₂OH)—CO—) can be replaced by the partial structure—CH₂—CH₂—CO— in aminopentanoic acid, the 2nd serine residue acts atleast as a spacer for separating amino-terminal amino group of ghrelinby a predetermined distance from the octanoyl group at the 3rd position.The reason that the activity was maintained by replacement of the 2ndserine residue by 5-aminopentanoic acid is that the basicity of theamino-terminal was increased by introducing its alkylamine structure.

In summary, the amino group of amino-terminal glycine residue isconsidered to confer basicity on the amino-terminal of ghrelin molecule,thus expressing the activity of ghrelin, and therefore the amino groupat the amino-terminal is preferably not blocked.

Further, the 2nd serine residue is considered to act as a spacer forseparating the amino group at the amino-terminal by a predetermineddistance from the octanoyl group at the 3rd position, and therefore the2nd serine residue may be replaced by an amino acid or non-amino acidcompound having a relatively less bulky side chain. That is, theposition of the octanoyl group in the ghrelin molecule is definedrelative to the amino-terminal amino group, and this positionalrelationship constitutes a part of the active structure of ghrelin.

That is, the side chain of amino acid 2 is preferably relatively lessbulky such as in serine, alanine and norvaline rather than an amino acidhaving a bulky structure, and an amino acid residue not restricting theflexibility of neighboring residues is preferable as the amino acid 2.Further, because the Ca-increasing activity ofN^(α)-aminopentanoyl-ghrelin (3-7) amide is almost maintained (EC₅₀=3.4nM), 2nd serine can be replaced by a non-amino acid compound.

(5) Configuration of Amino Acid Residues 3 and 4

On the basis of the structure of ghrelin(1-7) amide, its derivativeswherein 3rd L-serine and 4th L-phenylalanine had been replaced by thecorresponding D-amino acids were prepared, and the influence which theconfiguration of amino acid 3 and 4 have on the Ca-releasing activitywas examined. Specifically, on the basis of [serine³ (octyl)]-ghrelin(1-7) amide (EC₅₀=5.8 nM) [Compound 50 in Table 11] and [cysteine³(octyl)]-ghrelin (1-7) amide (EC₅₀=7.4 nM) [Compound 48 in Table 11]which maintained a good activity, their derivatives wherein 3rd serineand 4th phenylalanine had been replaced by the corresponding L- orD-amino acids were prepared.

The results are summarized in Table 11. From these results, both aminoacids 3 and 4 are preferably L-amino acids.

TABLE 11 Ghrelin derivative activity 8 Ca-releasing Compound activitystructure EC₅₀ (nM) 48. [Cys³(Octyl)]-Ghrelin (1-7)-amide 7.4H-Gly-Ser-Cys(C₈H₁₇)-Phe-Leu-Ser-Pro-NH₂ 49. [Cys³(Octyl),^(D)Phe⁴]-Ghrelin (1-7)-amide 3,000H-Gly-Ser-Cys(C₈H₁₇)-^(D)Phe-Leu-Ser-Pro-NH₂ 50. [Ser³(Octyl)]-Ghrelin(1-7)-amide 5.8 H-Gly-Ser-Ser (C₈H₁₇)-Phe-Leu-Ser-Pro-NH₂ 51.[Ser³(Octyl), ^(D)Phe⁴]-Ghrelin (1-7)-amide 2,200H-Gly-Ser-Ser(C₈H₁₇)-^(D)Phe-Leu-Ser-Pro-NH₂ 52.[^(D)Ser³(Octyl)]-Ghrelin (1-7)-amide >10,000 H-Gly-Ser- _(D)Ser(C₈H₁₇)-Phe-Leu-Ser-Pro-NH₂ 53.[^(D)Ser³(Octyl), ^(D)Phe⁴]-Ghrelin(1-7)-amide >10,000 H-Gly-Ser-^(D)Ser(C₈H₁₇)-^(D)Phe-Leu-Ser-Pro-NH₂

(6) Mode of Linkage of a Side Chain at the 3rd Position

Derivatives of ghrelin wherein the original ester linkage was replacedby an ester in the reverse direction (Compound No. 54), an amide(Compound Nos. 55 and 56), a disulfide (Compound No. 57) and methylene(Compound No. 58) were prepared such that the side chain at the 3rdposition became the same length as that of the ghrelin chain(—CH₂—O—CO—C₇H₁₅). In addition, ester derivatives having sterichindrance on the β-carbon atom of amino acid at the 3rd position(Compound Nos. 59 and 60) and an amide derivative wherein 3 methyleneunits had been extended (Compound No. 61) were prepared. The results aresummarized in Table 12.

TABLE 12 Ghrelin derivative activity 9 Activity Compound structure EC₅₀(nM) 54. [Asp³(O-Heptyl)]-hGhrelin 5.1GSD(O—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR 55. [Asp³(NH-Heptyl)]-hGhrelin 11GSD(NH—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR 56. [Dap³(Octanoyl)]-hGhrelin 2.6GS—NH—^(L)CH(CH₂NHCO—C₇H₁₅)—CO-FLSPEHQRVQQRKESKKPPAKLQPR 57.[Cys³(S-Heptyl)]-hGhrelin 1.4 GSC(S—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR 58.[Adod³]-hGhrelin 0.91 GS—NH—CH(n-C₁₀H₂₁)—CO-FLSPEHQRVQQRKESKKPPAKLQPR59. [Thr³(Octanoyl)]-hGhrelin 10 GST(CO—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR60. [Leu², Thr³(Octanoyl)]-hGhrelin 46GLT(CO—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR 61. [Lys³(Octanoyl)]-hGhrelin 32GSK(CO—C₇H₁₅)FLSPEHQRVQQRKESKKPPAKLQPR

The activity of Compound 58, wherein the side chain at the 3rd positionhad been replaced by methylene units exclusively, showed the strongestactivity (EC₅₀ value=1 nM or less). The activity of other derivativeswas varied depending on the type of linkage, but it was confirmed thatthe mode of linkage of a side chain of amino acid 3 does not exert asignificant influence on the activity.

(7) Hydrophobicity of a Side Chain at the 3rd Position

Derivatives wherein Ser (octanoyl) group 3 had been replaced by ahydrophobic amino acid most of which are natural amino acids wasprepared, and their activity was examined. The results are summarized inTable 13.

TABLE 13 10. Activity of Ghrelin derivatives Compound Activity StructureEC₅₀ (nM) 62. (Trp³)-hGhrelin 31 GSWFLSPEHQRVQQRKESKKPPAKLQPR 63.[Phe³]-hGhrelin 2,000 GSFFLSPEHQRVQQRKESKKPPAKLQPR 64. [Cha³]-hGhrelin19 GS-Cha-FLSPEHQRVQQRKESKKPPAKLQPR 65. [2-^(L)Nal³]-hGhrelin 8.2GS-^(L)Nal-FLSPEHQRVQQRKESKKPPAKLQPR 66. [2-^(D)Nal³]-hGhrelin >10,000GS- _(D) Nal-FLSPEHQRVQQRKESKKPPAKLQPR 67.[Ser³(Bzl)]-hGhrelinGSS(CH₂—C₆H₅) 7.6 FLSPEHQRVQQRKESKKPPAKLQPR 68.(Cys³(Trityl)]-hGhrelinGSC(C—Ph₃) 20 FLSPEHQRVQQRKESKKPPAKLQPR 69.[Ser³(4-Methylpentanoyl)]-hGhrelin 4.4 GSS(CO—CH₂CH₂CH(CH₃)₂)FLSPEHQRVQQRKESKKPPAKLQPR 70. [Leu³]-hGhrelin 4,400GSLFLSPEHQRVQQRKESKKPPAKLQPR 71. [Ile³]-hGhrelin >10,000GSIFLSPEHQRVQQRKESKKPPAKLQPR 72. [Lys³]-hGhrelin 120GSKFLSPEHQRVQQRKESKKPPAKLQPR 73. [Nle³]-hGhrelin 2,800GS-Nle-FLSPEHQRVQQRKESKKPPAKLQPR 74. [Val³]-hGhrelin 1,600GSVFLSPEHQRVQQRKESKKPPAKLQPR

The EC₅₀ values of the derivatives having an aromatic hydrophobic aminoacid such as tryptophan, cyclohexyl alanine or naphthyl alanine at the3rd position were 31 nM, 19 nM and 8.2 nM respectively, indicating thatthe Ca-releasing activity was maintained. Unexpectedly, when phenylalanine was introduced at the 3rd position, the Ca-releasing activitywas slightly low, but even if more hydrophobic Ser (Bzl) or Cys (Trityl)was introduced at the 3rd position, the Ca-releasing activity wassimilarly maintained, and thus it was confirmed that the hydrophobicityof the side chain at the 3rd position is more preferable for expressingthe activity.

On one hand, when an aliphatic hydrophobic amino acid such as leucine,isoleucine, norleucine or valine was introduced at the 3rd position, theCa-releasing activity of the derivatives was generally maintained butslightly lower than the derivatives introducing the aromatic amino acid.The activity of Compound 73 having norleucine at the 3rd position wasEC₅₀=2,800 nM, whereas the activity of 6-amino-norleucine (lysine;Compound 72) having an amino group added to a side chain of norleucinewas increased to 120 nM in terms of EC₅₀ values, so it was confirmedthat similar to the basicity of carboxyl-terminal described above, thebasicity of a side chain at the 3rd position is also preferable

(8) Short-Chain Ghrelin Derivatives

As described above, it was found that a ghrelin fragment ofamino-terminal amino acids 1 to 4 shows significant activity and thisactivity is further increased by adding leucine at 5th position to saidfragment; 3rd amino acid residue is preferably the one having ahydrophobic side chain; the activity is increased by introducing a basicresidue; and amino acid residues 1 and 2 may be replaced by a non-aminoacid compound having a 2-residue length, such as δ-amino acid. On thebasis of these results, various short-chain ghrelin derivatives based onthe amino-terminal region (1-5) were prepared as shown in Compound Nos.76 to 87 in Tables 14 and 15, and their activities were examined. Theresults are summarized in Tables 14 and 15.

Compound 80 is known (Ipamorerin; K. Raum et al., Eur. J. ofEndocrinol., 139: 552-561, 1998).

TABLE 14 Ghrelin derivative activity 11 Activity Compound structure EC₅₀(nM) 75. (Lys⁷]-Ghrelin (1-7)-amide 11H-Gly-Ser-Ser(CO—C₇H₁₅)-Phe-Leu-Ser-Lys-NH₂ 76. 12 [N-Aminopentanoyl,Ser³(Octyl), Lys⁵]-Ghrelin (3-5)-amideNH₂—(CH₂)₄—CO-Ser(C₇H₁₇)-Phe-Lys-NH₂ 77. [N-Aminopentanoyl,^(D)Ser³(Octyl),^(D)Phe⁴, Lys⁵)- 1,600 Ghrelin (3-5)-amideNH₂—(CH₂)₄—CO-^(D)Ser(C₈H₁₇)-^(D)Phe-Lys-NH₂ 78. [Aib¹, His²,Ser³(Octyl), Lys⁵]-Ghrelin (1-5)-amide 34H-Aib-His-Ser(C₈H₁₇)-Phe-Lys-NH₂ 79. 38 [Aib¹, His², ^(D)Ser³(Octyl),^(D)Phe⁴, Lys⁵]-Ghrelin (1-5)-amide H-Aib-His-^(D)Ser(C₈H₁₇)-^(D)Phe-Lys-NH₂ 80. [Aib¹, His², ^(D)Nal³, ^(D)Phe⁴,Lys⁵]-Ghrelin (1-5)-amide 2.5 H-Aib-His-^(D)Nal-^(D)Phe-Lys-NH₂

Since the Ca-releasing activity of known Compound 80 was high (2.5 nM),the activity of Compound 79 derived from Compound 80 by replacing2-D-naphthyl alanine at the 3rd position by D-octyl serine was alsoexamined, and as a result, its EC₅₀ value was 38 nM, indicating that theactivity was maintained. Compound 77 having D-octyl serine and D-phenylalanine at the 3rd and 4th positions, which has the same amino acidstructure as that of Compound 79 except for amino acids 1 and 2, showeda lower activity (1,600 nM), and these results indicates that thesequence or structure of amino acids 1 and 2 also affects the stericconfiguration of side chains of 3rd and 4th amino acids important forexhibiting the activity.

That is, in the case where amino acids 1 and 2 were replaced byaminopentanoic acid, the activity was kept at 34 nM even if 2-D-naphthylalanine at the 3rd position and D-phenyl alanine at the 4th positionwere replaced by their corresponding L-amino acids (Compound 78), andthus the amino acid sequence (Gly-Ser) at the 1st to 2nd positions inghrelin requires L configuration for amino acids 3 and 4, but even ifamino acids 3 and 4 have D configuration, the activity is madesignificant by introducing another amino acid sequence such as Aib-His.It was also confirmed that regardless of L- or D-configuration at the3rd and 4th positions, the activity is expressed by introduction ofaminopentanoic acid at the 1st and 2nd positions.

TABLE 15 Ghrelin derivative activity 12 Compound Activity Structure EC₅₀(nM) 81. [N-Aminopentanoyl, Ser³(Octyl)]-Ghrelin (3-5)-amide 11NH₂—(CH₂)₄—CO-Ser(C₈H₁₇)-Phe-Leu-NH₂ 82. (N-Aminopentanoyl,Ser³(Octyl)]-Ghrelin 12 (3-5)-methylamideNH₂—(CH₂)₄—CO-Ser(C₈H₁₇)-Phe-Leu-NH—CH₃ 83. 22 [N-Aminopentanoyl,Ser³(Octyl)]-Ghrelin (3-5)-ethylamide NH₂—(CH₂)₄—CO-Ser(C₈H₁₇)-Phe-Leu-NH—C₂H₅ 84. [N-Aminopentanoyl, Ser³(Octyl) [-Ghrelin 98(3-5)-benzylamide NH₂—(CH₂)₄—CO-Ser (C₈H₁₇)-Phe-Leu-NH—CH₂—C₆H₅ 85.[N-Aminopentanoyl, Ser³(Octyl)] 3.5 -Ghrelin (3-5), aminoethylamide NH ²—(CH₂)₄—CO-Ser(C₈H₁₇)-Phe-Leu-NH—(CH₂)₂—NH₂ 86. [N-Aminopentanoyl,Ser³(Octyl), MePhe⁴, MeLeu⁵]- 82 Ghrelin (3-5)-amide NH₂—(CH₂)₄—CO-Ser(C₈H₁₇)—MePhe-MeLeu-NH₂ 87. [^(D)Leu⁵]-hGhrelin 220 GSS(CO—C₇H₁₅)F-^(D)L-SPEHQRVQQRKESKKPPAKLQPR

Using [N-Aminopentanoyl, Ser³(Octyl)]-Ghrelin (3-5) based on theamino-terminal region (1-5) of ghrelin, the correlation between activityand structure in the carboxyl-terminal region was examined. The activityof its derivatives wherein carboxyl-terminal leucine at the 5th positionhad been modified with amide, methyl amide, ethyl amide or benzyl amidewas maintained but tended to be decreased as shown by EC₅₀ values of 11nM, 12 nM, 22 nM and 98 nM, respectively. On the other hand, byreplacing ethyl amide by aminoethyl amide, the activity was increased asshown by an EC₅₀ value of 3.5 nM, thus revealing that impartment ofbasicity to the carboxyl-terminal of ghrelin molecule is preferable.

These various carboxyl-terminal amide derivatives are useful compoundsbecause of their resistance in vivo to decomposition with carboxypeptidases. Compound 86 (EC₅₀=86 nM) containing N-methylamino acid isalso an useful compound because of its resistance to the enzymes.

Example 12 GH Releasing Activity of Ghrelin Derivatives in Rat (1)GH-Releasing Activity of Various Long-Chain Ghrelin Derivatives in Rat

18 nmol/kg Compound 17 ([Ser³(Octyl)]-hGhrelin), 30 nmol/kg Compound 18([Cys³(Octyl)]-rGhrelin), 100 nmol/kg Compound 65([2-^(L)Nal³]-hGhrelin), or 18 nmol/kg Compound 15([Ser³(3-Phenylpropinyl)]-hGhrelin) was administered rapidly andintravenously into IGS-SD strain rats (about 7-week-old) underanesthesia with Nembutal for each sample (n=3). Fifteen minutes afteradministration, plasma was collected, and the concentration of GH inplasma was measured by radioimmunoassay (Biotrak/Amersham). Separately,0.2% bovine serum albumin (BSA)-physiological saline, 6 nmol/kg rGhrelinand hGhrelin, or 80 nmol/kg Ipamorelin (Compound 80) was administeredinto other rats as the control, respectively, and the concentrations ofGH in plasma in 15 minutes after administration were compared (for eachsample, n=3).

The results are shown in Table 13. Compound 17 ([Ser³(Octyl)]-hGhrelin),Compound 18 ([Cys³ (Octyl)]-rGhrelin) and Compound 15([Ser³(3-PhPrl)]-hGhrelin) exhibited a strong GH-releasing activity, andthe GH-releasing activity of [2-^(L)Nal³]-hGhrelin showed a goodcorrelation with intracellular Ca-releasing activity.

TABLE 16 GH-releasing activity of various long-chain ghrelin derivativesGH level in plasma EC₅₀ in 15 min. after Compound value doseadministration (ng/mL) administered (nM) (nmol/kg) Rat 1 Rat 2 Rat 3 M ±S.D. Physiological — — 32 52 59  49 ± 12 saline hGhrelin 1.3 6 1802 16132203 1873 ± 301 rGhrelin 1.5 6 2056 1082 1205 1448 ± 530 Ipamorelin 2.580 377 260 1184  607 ± 503 (Compound 80) [Ser³(Octyl)]- 1.2 18 1626 16021743 1657 ± 75  hGhrelin [Cys³(Octyl)]- 5.4 30 2786 2342 2354 2494 ± 253rGhrelin [Ser³(Phenyl- 1.4 18 2119 2078 1581 1926 ± 299 propionyl)]-hGhrelin [2-^(L)Nal³]- 8.2 100 1637 1576 1357 1524 ± 147 hGhrelin

(2) Change of GH in Plasma by Administration of [Cys³ (Octyl)]-RatGhrelin

After Compound 18 ([Cys(Octyl)]-rat ghrelin) was intravenouslyadministered in a dose of 5 μg/head into Wistar strain male rats (about260 to 280 g) under anesthesia with Nembutal, GH released to blood wasmeasured. Physiological saline as the control and rat ghrelin (5μg/head) were also administered and compared with Compound 18.

As shown in Tables 17 to 19, the GH secretion-promoting activity of[Cys³ (Octyl)]-rat ghrelin was equivalent to natural rat ghrelin (thatis, C_(max) of secreted GH was about 1,100 ng/ml for both ghrelins), andfurther the secretion time tended to prolong. The intracellularCa-releasing activity of Compound 18 was 5.4 nM in terms of EC₅₀.

TABLE 17 Change of GH level in plasma by administration of[Cys³(Octyl)]-rat ghrelin [Cys(C18)³]- Time rat ghrelin (min) 5 μg/head0 5 10 15 20 30 60 GH Rat 1 377 338 687 927 900 469 98 level Rat 2 101294 258 300 358 245 86 in Rat 3 59 476 949 1229 1417 704 133 plasma Rat4 33 530 959 1451 1299 800 220 (ng/mL) Rat 5 32 613 1060 1561 1359 726122 Mean ± S.D. 120 ± 146 450 ± 133 783 ± 324 1093 ± 506 1067 ± 445 589± 229 132 ± 53

TABLE 18 Change of GH level in plasma by administration of physiologicalsaline Physiological Time (min) saline 0 5 10 15 20 30 60 GH Rat 1 0 88129 133 116 107 430 level Rat 2 204 122 118 134 128 69 36 in Rat 3 77 00 0 0 0 11 plasma Rat 4 0 0 0 0 48 27 110 (ng/mL) Rat 5 0 0 0 0 0 0 210Mean ± S.D. 56 ± 89 42 ± 58 49 ± 67 53 ± 73 58 ± 61 41 ± 47 159 ± 170

TABLE 19 Change of GH level in plasma by administration of rat ghrelinRat ghrelin Time (min) 5 μg/head 0 5 10 15 20 30 60 GH level Rat 1 143186 425 405 215 56 3 in Rat 2 10 1396 2028 1566 876 242 27 plasma Rat 3838 163 443 681 419 120 36 (ng/mL) Rat 4 348 556 1387 1469 1293 663 100Rat 5 0 875 1380 1009 1414 452 20 Mean ± S.D. 268 ± 348 635 ± 517 1133 ±690 1026 ± 498 843 ± 525 306 ± 250 37 ± 37

Example 13 Increasing Action of Ghrelin on Appetite

(1) Appetite-Increasing Action by Administration into Ventricle

Physiological saline containing rat ghrelin at various concentrationswas administered at 8:45 a.m. into cerebral ventricles of male Wistarstrain rats (16 to 20 animals per group) each weighing 300 to 325 g. Asthe control, ghrelin-free physiological saline was administered intoventricles. After administration, the rats were allowed feed ad libitum,and the amount of feed taken for 2 hours after administration wasmeasured. As shown in FIG. 6, an increase in the amount of feed takenwas observed in the rats administered 50 μmol ghrelinintracerebroventricularly, and a dose-dependent increase in the amountof feed taken was observed in the rat administered 200 pmol and 500 pmolghrelin, but the amount of feed taken was decreased in the ratsadministered 2 nmol ghrelin. Usually, the rat takes feed at night, sothat in the morning, the rat is on a full stomach and rarely takes feed(see the rat administered physiological saline as the control in FIG.6), and thus the increase in the amount of taken feed by administratingghrelin into cerebral ventricle indicates that ghrelin has anappetite-increasing action.

(2) Appetite-Increasing Action by Intravenous Administration

50 μg/kg rat ghrelin was intravenously administered into tail veins in9-month-old male SD (Sprague-Dawley) rats (5 animals) and Wister rats (4animals), and the amount of feed taken for 2 hours after administrationwas measured (evaluated during 16:00 to 19:00 p.m.). As shown in Table20, the amount of feed taken was evidently increased by intravenousadministration of ghrelin, as compared with the amount of feed takenwithout administration of rat ghrelin, which was determined using thesame animal at the same hour on another day. That is, it wasdemonstrated that ghrelin has an appetite-increasing action even byintravenous administration.

TABLE 20 Amount of feed taken (g) No Administration administrationstrain Rat No. of ghrelin of ghrelin S-D 1 3.2 2.2 2 3.7 1.0 3 3.2 0.1 42.7 1.3 5 2.6 0.8 Mean 3.1 1.1 S.D. 0.4 0.8 Wister 6 2.3 0.2 7 1.9 1.4 81.6 0.1 9 2.1 0.3 Mean 2.0 0.5 S.D. 0.3 0.6

Example 14 Enhancement of Gastric Functions by Ghrelin

To examine the effect of ghrelin on gastric functions, the followingexperiment was carried out. Male SD strain rats (7- to 8-week-old,weighing 200 to 280 g) were fasted for 20 hours or more and then used inthe experiment. The rats were anesthetized by intraperitonealadministration of urethane (1.25 g/kg) and kept warm using a warming padand a warming light. A tracheal canula was inserted, and the esophaguswas ligated by silk thread, and each rat was subjected to the followingoperation in order to measure gastric acid secretion or gastricmotility. In the experiment using conscious animal, the rat wassubjected to the operation for measurement of gastric acid secretion orgastric mobility under slight anesthesia by inhalation of ether.

In the experiment for gastric acid secretion under anesthesia withurethane, the operation was conducted according to the method proposedby Ohno et al. [Ohno, T., et al., Jpn. J. Pharmacol. 43, 429-439(1987)]. Briefly, in the supine position the abdomen was incised and thestomach and duodenum were exposed. A polyethylene tube was inserted intoa front part of the stomach to prepare acute stomach fistula. Anotherpolyethylene tube was inserted into the stomach after cleaving theduodenum, and the surrounding part of the pylorus was ligated and fixed.The inside of the stomach was infused with physiological saline whichwas adjusted to pH 7.0 in a reservoir and warmed at 37° C. The flow ratewas 1.0 ml/min. The infusion fluid was adjusted to pH 7.0 by titrationwith 100 mM NaOH using a pH-fixing unit (Hirariuma, Comitite-8). Afterit was confirmed that a basic amount of gastric acid secretion wasstable, the test chemical was intravenously administered, and the rateof secretion of gastric acid was measured at 5-minute intervals. Fourrats were used in each group.

In the experiment during arousal, the rat was subjected to the sameoperation under slight anesthesia by inhalation of ether, and then asmall cut was made in the flank, and an infusion tube was taken out fromthe body. The exposed stomach and duodenum were put back in the abdomen,and the excised site was sutured, and the animal was fastened whilelying on the back in a Ballman-type rat-fixing cage, and after it wasconfirmed that the rat was recovered from anesthesia, the rat wassubjected to the experiment. The esophagus was ligated, but a tracheacanula was not inserted.

The experiment for measurement of stomach motility under urethaneanesthesia, a miniaturized balloon method was used according to themethod proposed by Takeuchi & Nobuhara [Takeuchi, K. and Nobuhara, Y.,Digestive Diseases and Sciences 30, 1181-1188 (1985)]. That is, aballoon filled with water and a supporting catheter were inserted intothe stomach after cleavage of a front part of the stomach. It was fixedto lie on a gland of the stomach line, and one end of the catheter wasconnected to a pressure transducer (LPU-0.1-350-0-II, from Nihon KohodenCorporation). After it was confirmed that the gastric motility wasstable, the test chemical was intravenously administered accumulativelyat 60-minute intervals. For the gastric motility, the amplitude ofinternal pressure in the stomach and the number of shrinking reactionsin shrinkage motility having an amplitude of 20 cm H₂O or more weremeasured at 10-minute intervals. Four animals was used in each group. Inthe experiment using conscious animals, the rat was subjected to thesame operation under light anesthesia by inhalation of ether, and afterthe excised site was sutured, the animal was fastened in the proneposition in a Ballman-type rat-fixing cage. After it was confirmed thatthe rat was recovered from anesthesia, the animal was subjected to theexperiment.

Rat ghrelin and histamine dihydrochloride were dissolved inphysiological saline and administered in a dose of 1 ml/kg into tailvein. To examine whether the vagus nerve action is involved in theaction of ghrelin, atropine sulfate was subcutaneously administered in30 minutes before administration of ghrelin, or the cervical vagus nervebundles were bilaterally cut off. To examine the involvement ofhistamine H₂ receptor in the action of ghrelin, famotidine (Gaster®,produced by Yamanouchi Pharmaceutical Co., Ltd.) was subcutaneouslyadministered 30 minutes before administration of ghrelin. The resultsare shown in mean±standard error. Statistical analysis was performedusing Dunnett' s multiple comparison tests. P value <0.05 was judged tobe statistically significant.

As shown in FIG. 7A and in Table 21, secretion of gastric acid waspromoted in a dose-dependent manner upon intravenous administration ofrat ghrelin in a dose of 0.8 to 20 μg/kg into the rat under urethaneanesthesia.

In the rat under anesthesia, the spontaneous motility of stomach washardly observed before administration of ghrelin. When rat ghrelin wasintravenously administered in a dose of 0.8 to 20 μg/kg into the rat inthis condition, both the amplitude and frequency of gastric motilitywere promoted as shown in FIGS. 8A & B and in Table 21. These reactionswere observed immediately after administration of rat ghrelin. Byadministration of 20 μg/kg, secretion of gastric acid was increased andreached to the maximum level within 20 minutes and gradually decreasedfor 90 minutes after administration. As shown in FIGS. 7 A & B, themaximum reaction in the gastric acid secretion-promoting action byadministration of 20 μg/kg rat ghrelin was almost comparable to thereaction induced by intravenous administration of 3 mg/kg histamine. Theaction of promoting the amplitude of gastric motility reached themaximum reaction within 10 minutes in any dose, and by administration of20 μg/kg ghrelin, the action was gradually decreased until 50 minutesafter administration.

Further, as shown in Table 21, the action of promoting gastric secretioninduced by administration of 20 μg/kg rat ghrelin was inhibited almostcompletely by pretreatment with atropine or bilateral cervical vasotomy,but this action was not affected by pretreatment of subcutaneousadministration of 1 mg/kg famotidine i.e. a histamine H₂ receptorantagonist. Further, the action of promoting gastric motility induced byadministration of rat ghrelin was completely inhibited by pretreatmentwith atropine or bilateral cervical vagotomy. From these results, it wasconfirmed that the promoting action of ghrelin on gastric functions isnot via histaminergic mechanism but via activation of vagus nervesystem.

By intravenous administration of rat ghrelin (4 and 20 μg/kg), secretionof gastric acid was promoted in the conscious rat in the same way as inthe rat under urethane anesthesia. As compared with the rat underanesthesia, the conscious rat had spontaneous gastric motility beforeadministration of the test chemical, and even in this condition byadministering 0.8 to 20 μg/kg rat ghrelin into the rat, the gastricmotility was promoted together with its amplitude and frequency. Fromthe above result, it was confirmed that by intravenous administration ofghrelin, promotion of gastric acid secretion and promotion of gastricmotility occur not only in the anesthetized rat but also in theconscious rat.

TABLE 21 Gastric acid secretion Gastric motility (μ Frequency Amplitudeequivalent/ (times/ (cm H₂O/ Treatment 60 min) 60 min.) 60 min.)Physiological saline 17.6 ± 1.2 1.3 ± 1.0  1.7 ± 1.0 Rat ghrelin 0.8μg/kg 24.5 ± 2.2 35.5 ± 18.1  6.7 ± 4.4 i.v. injection 4 μg/kg 23.5 ±2.6 60.8 ± 25.6 11.1 ± 5.3 i.v. injection 20 μg/kg 43.3 ± 4.6 100.5 ±20.4  21.8 ± 2.5 i.v. injection (*1) (*1) (*1) Intravenous + atropine26.1 ± 3.9 0 0 injection of 1 mg/kg (*2) (*3) (*3) 20 μg/kg ratsubcutaneous ghrelin administration + removal of 18.4 ± 3.7 0 0 vagusnerve (*3) (*3) (*3) + famotidine 43.0 ± 4.2 NT NT 1 mg/kg subcutaneousadministration Symbols in the table indicate: *1, p < 0.01; *2, p <0.05; and *3, p < 0.01 NT: Not tested.

Example 15 Promoting Action of Ghrelin and Ghrelin Derivatives on CellGrowth

To examine the action of administered ghrelin on promotion of cellgrowth, the following experiment was conducted. Twenty μg/kg of ratghrelin or thioether-type rat ghrelin (Compound 18[Cys³(octyl)]-hGhrelin) was administered into tail veins of Wister malerats (7.5-week-old) respectively. Seventeen hours after administration,³H-thymidine was administered into tail veins, and 1 hour thereafter,duodenum, jejunum and bone marrow were excised. The incorporation of³H-thymidine to DNA fractions of these tissues was measured in order toexamine the cell growth-promoting action of ghrelin and ghrelinderivatives. The tissues were cut thin and homogenized using a Polytronhomogenizer, and after centrifugation, the supernatant was precipitatedwith trichloroacetic acid to give a DNA fraction. The radioactivity ofthe DNA fraction was measured by a liquid scintillation counter.

As shown in Table 22, the incorporation of ³H-thymidine into thesetissues or organs was increased by intravenous administration of ratghrelin or thioether-type rat ghrelin, and it was thus confirmed thatghrelin exhibits a cell growth-promoting action in duodenum, jejunum andbone marrow.

The time course of the cell growth-promoting action after intravenousadministration of ghrelin was similar to that after administration ofGHRH (growth hormone releasing hormone), so it was considered that thecell growth-promoting action of ghrelin occurs via GH (growth hormone)secreted mainly from pituitary. It was considered that the regulation ofGH secretion by ghrelin as a physiological factor is reasonable fororganism regulation, and there are less adverse effects which couldoccur by GH administration.

TABLE 22 Comparative Rat Thioether-type Example ghrelin ghrelin Bonemarrow 100.0 ± 141.7 ± 144.5 ± (in 17.8% 30.1% 16.5% tissues) Duodenum100.0 ± 136.0 ± 114.0 ± (in DNA 14.2% 17.8% 11.7% fraction) Jejunum100.0 ± 159.0 ± 151.0 ± (in DNA 6.8%  7.5%  23.6% fraction)Numerical values show the ratio (%) of incorporation of radioisotoperelative to the mean (in triplicate) of the comparative example (i.e.the group given physiological saline).

Example 16 Quantification of Ghrelin by Anti-Ghrelin Antibody

Using antibodies raised against amino- and carboxyl-terminal rat ghrelinpeptides as antigens, ghrelin in various living tissues was quantifiedby radioimmunoassay (RIA).

Rabbits were immunized with [C-Cys]-rat ghrelin [1-11] (rat ghrelinpeptide of amino acids 1 to 11 from amino-terminal having cysteine boundto the carboxyl-terminal thereof) and [C-Cys]-rat ghrelin [13-28] (ratghrelin peptide of amino acids 13 to 28 from amino-terminal havingcysteine bound to the carboxyl-terminal thereof) as antigens, to formamino-terminal antibody (anti-[C-Cys]-rat ghrelin [1-11] antigen) andcarboxyl-terminal antibody (anti-[C-Cys]-rat ghrelin [13-28] antigen)respectively.

As shown in FIG. 9 a, the IC₅₀ (50% inhibitory concentration) of ratghrelin was 3.1 fmol in binding between radioisotope-labeled rat ghrelinand the amino-terminal antibody. This amino-terminal antibody, whileshowing 100% cross-reactivity with chemically synthesized human ghrelinand rat ghrelin, showed only 0.3% cross-reactivity with n-hexanoyl ratghrelin wherein 3rd serine had been modified with n-hexanoyl group and20% cross-reactivity with n-decanoyl rat ghrelin wherein 3rd serine hadbeen modified with n-decanoyl group. Further, the amino-terminalantibody did not react with ghrelin from which fatty acid had beenreleased.

The amino-terminal antibody showed similar affinity for rat ghrelin (28amino acids), human ghrelin (28 amino acids), and ghrelin-27 (ghrelinconsisting of 27 amino acids) found in human and rat. Accordingly, itwas confirmed that the amino-terminal antibody specifically recognizesnatural ghrelin wherein 3rd serine was modified with n-octanoyl group.

As shown in FIG. 9 b, natural rat ghrelin modified with n-octanoyl groupand rat ghrelin modified by removing n-octanoyl from the natural ratghrelin showed a similar IC₅₀ value of 44 fmol in binding betweenradioisotope-labeled rat ghrelin and the carboxyl-terminal antibody.That is, it was confirmed that the carboxyl-terminal antibody has thesame affinity for ghrelin modified with fatty acid and for ghrelin fromwhich fatty acid was released.

These results revealed that in regard to ghrelins occurring in varioustissues in a living body, ghrelin wherein 3rd serine was modified withn-octanoyl group can be quantified by the amino-terminal antibody, whileboth ghrelin modified with fatty acid and ghrelin from which fatty acidwas released can be quantified by the carboxyl-terminal antibody.

Table 23 shows the result of examination of the contents of fattyacid-modified ghrelin and the contents of both fatty acid-modifiedghrelin and fatty acid-released ghrelin in various tissues in a livingbody.

TABLE 23 In the table, C-RIA indicates the result of quantification byradioimmunoassay using the carboxyl-terminal antibody, while N-RIAindicates the result of quantification by the amino-terminal antibody.Amount of rat ghrelin reacting with antibody (fmol/mg tissues) TissuesC-RIA N-RIA Hypothalamus 1.8 ± 0.3 <0.05 Pituitary 8.5 ± 3.1 <0.05Thyroid 3.5 ± 2.0 <0.05 Mandible  8.8 ± 1.3 <0.05 gland Thymus 3.5 ± 0.4<0.05 Adrenal gland 3.1 ± 0.4 <0.05 Atrium 2.3 ± 0.2 0.07 ± 0.01Ventricle 2.1 ± 0.1 <0.05 Aorta 2.4 ± 0.7 0.14 ± 0.03 Lung 3.1 ± 0.4<0.05 Liver 2.8 ± 0.5 <0.05 Pancreas 2.6 ± 0.6 0.15 ± 0.05 Stomach1779.8 ± 533.9  377.31 ± 55.83  Duodenum 106.7 ± 7.3  20.57 ± 0.69 Jejunum 60.2 ± 17.2 10.73 ± 5.44  Ileum 20.5 ± 5.1  0.16 ± 0.08 Cecum15.1 ± 2.5  1.70 ± 5.44 Colon 10.4 ± 0.7  <0.05 Kidney 5.4 ± 0.3 <0.05Spermary 2.8 ± 0.2 <0.05 Plasma (1 mL) 219.6 ± 71.8  4.02 ± 1.91

The numerical values in the table indicate “mean±standard deviation”.

Example 17 Production of Rat Ghrelin (1-28) by a Semi-Synthesis MethodSynthesis Scheme

In this example, rGhrelin was produced from rGhrelin (6-28) and Ghrelin(1-7) fragments previously prepared by genetic engineering method andchemical synthesis respectively, as follows.

Specifically, β-galactosidase 97S-(QFE-SRHRR)-rGhrelin (6-28), that is,a fusion protein of β-galactosidase 97S and rGhrelin (6-28) betweenwhich an amino acid sequence (-QFE-SRHRR-) having a site cleaved by V8protease and KexII protease occurred was expressed in E. coli. Thisfusion protein was treated with V8 protease, to cut off SRHRR rGhrelin(6-28). Then, all amino groups of SRHRR rGhrelin (6-28) were protectedwith Boc groups, and the resulting peptide was treated with KexIIprotease, to give [Lys (Boc)^(11, 16, 19, 20, 24)]-Ghrelin (6-28) fromwhich the amino-terminal amino group of Ser 6 had been isolated. Thisprotected fragment was condensed with [N^(α)-Boc]-rGhrelin (1-5)-Osuobtained by chemical synthesis, and the resulting[Lys(Boc)^(11, 16, 19, 20, 24)]-rGhrelin was treated with an acid,whereby rGhrelin was produced.

In this example, semi-synthesis of rGhrelin was described, but hGhrelincan also be synthesized by this method.

Further, in this example, fragment (1-5) was condensed with fragment(6-28), but chemically synthesized amino-terminal fragments (1-2), (1-3)and (1-7) can be condensed respectively with carboxyl-terminal fragments(3-28), (4-28) and (8-28) of an arbitrary length consisting of aminoacids at the 28th position up to the 3rd position constructed by geneticengineering means, in order to produce ghrelin as a fusion protein. Toreduce the number of steps in chemical synthesis, the condensationbetween (1-2) and (3-28) or between (1-3) and (4-28) is advantageous.From the viewpoint of complete prevention of the racemization caused bycondensation, the condensation between (1-7) and (8-28) by using Pro 7is particularly preferable.

Construction of Expression Vector pG97s rGR and Expression of Ghrelin(6-28) as a Fusion Protein

On the basis of the nucleotide sequence of rat ghrelin cDNA, a DNAfragment for rGhrelin (6-28) having an amino acid sequence QFE-SRHRR inthe prepro region was obtained by annealing using a total syntheticoligomer.

To insert this DNA fragment into pG97SnPPH34 (JP-A 9-296000),pG97SnPPH34 was treated with SalI and SmaI thereby deleting its humanparathyroid hormone precursor gene. The product was treated with alkaliphosphatase and ligated by T4 ligase to the rGhrelin derivative genefragment previously treated with SalI and kinase. The ligated plasmidwas transformed into E. coli DH5α strain, to give plasmid pG97s rGR.

The resulting plasmid pG97s rGR was transformed into E. coli M25 (ompT),and the resulting transformant was cultured onto 3 dishes eachcontaining 200 ml Terrific broth liquid medium (1.2% trypton, 2.4% yeastextract, 0.4% glucose) and cultured under shaking at 37° C. When theconcentration (OD₆₆₀) of the bacterial cell reached 0.8, isopropyl1-thio-β-galactopyranoside (IPTG) was added thereto at a finalconcentration of 2 mM, to express rGhrelin (6-28) fusion protein.Further, the bacterial cell was cultured for 4 hours and then collectedby centrifugation. The structure of rGhrelin (6-28) fusion protein is asfollows:

rGhrelin 6-28 fusion protein: (β-Galactosidase-97S)-QFE-SRHRR rGhrelin(6-28)Processing of rGhrelin 6-28 Fusion Protein and Purification of[SRHRR]-rGhrelin (6-28)

20 ml of the resulting bacterial cell was suspended in TE buffer and thebacterial cell was disrupted by a French press. Thereafter, theinclusion body was collected by centrifugation at 3000 rpm for 15minutes, suspended again in 10 ml TE buffer and deionized water, andcentrifuged whereby the inclusion body was washed. The inclusion bodywas diluted with deionized water such that its OD₆₆₀ was reduced to50.0, and Tris-HCl (pH 8.2) was added thereto at a final concentrationof 50 mM, and the inclusion body was dissolved in urea (finalconcentration: 3.5 M). To this solution kept at 30° C. was added rV8protease derivative V8D5 (abbreviated herein after to V8D5) (JP-A9-47291) at a final concentration of 10 μg/ml, and the solution wastreated with the enzyme at 30° C. for 20 minutes. The reaction wasterminated by adding 3% acetic acid (AcOH).

1.5-fold excess deionized water was added to the V8D5 enzymereaction-terminated solution containing the [SRHRR]-rGhrelin (6-28),then this solution was adjusted to pH 5.0 with 5N NaOH, to precipitatethe β-galactosidase derivative fragment which was then removed bycentrifugation at 5000 rpm for 10 minutes.

The supernatant containing [SRHRR]-rGhrelin (6-28) was applied toTSK-ODS 80Ts column (resin particle diameter of 20 μm, 50 mm I.D.×100mm, TOSOH Co., Ltd.) previously equilibrated with 0.1% TFA. The desiredpeptide was eluted by a linear gradient of from 100% buffer A [0.8ml/min., 1% acetonitrile, 0.1% TFA] to 100% buffer B [50 acetonitrile,0.095% TFA], which was programmed to be finished in a volume of 5columns. Fractions containing the desired peptide [SRHRR]-rGhrelin(6-28) were collected (about 50 mg).

Purification of [Boc-SRHRR]-[Lys(Boc)^(11, 16, 19, 20, 24)]-rGhrelin(6-28)

6-equivalent mole (19.2 mg, 6×15 μmol) of di-t-butyl bicarbonate wasadded to 50% aqueous acetonitrile solution containing about 50 mg (15μmol) of [SRHRR]-rGhrelin (6-28), then adjusted to pH 9 withtriethylamine, and left at room temperature for 15 minutes. Acetic acidwas added at a final concentration of 0.5% to the reaction solution, andafter the acetonitrile was evaporated, the solution was added toEMPORE-Octyl (C8) HD 4 mm/1 ml cartridge previously equilibrated with 10acetonitrile containing 0.1% TFA, and after the column was washed withthe equilibration solution, [Boc-SRHRR]-[Lys(Boc)^(11, 16, 19, 20, 24)]-rGhrelin (6-28) was eluted with 90%acetonitrile containing 0.095% TFA. The acetonitrile was evaporated, and6 ml solution containing about 30 mg of the desired peptide wasobtained.

Mass spectrometry indicated mainly two peptides whose molecular weightafter Boc modification was higher by 500 (determined molecular weight,3895) or by 600 (determined molecular weight, 3995) than the molecularweight (determined molecular weight=3396, theoretical molecularweight=3398) before Boc modification.

Cleavage of [Lys (Boc)^(11, 16, 19, 20, 24)]-rGhrelin (6-28) by Kex2Protease and Purification Thereof.

A calcium chloride solution and Tris-HCl, pH 8.2, were added at finalconcentrations of 0.3 mM and 20 mM respectively to the resulting aqueoussolution of [Boc-SRHRR]-[Lys (Boc)^(11, 16, 19, 20, 24)]-rGhrelin (6-28)(30 mg, 6 mL). After a solution of Kex2 protease (JP-A 10-229884) wasadded thereto at a concentration of 1×10⁵ units/ml, the sample wastreated with the protease at 30° C. for 60 minutes.

In HPLC, a peak of [Boc-SRHRR]-[Lys (Boc)^(11, 16, 19, 20, 24)]-rGhrelin(6-28) disappeared, a peak of [Lys (Boc)^(11, 16, 19, 20, 24)]-rGhrelin(6-28) was shifted toward the side of hydrophobicity, and a peak of ahydrophilic fragment corresponding to Boc-SRHRR was observed.

After the disappearance of the starting material was confirmed, thereaction solution was adjusted to pH 3.5 with aqueous acetic acid andapplied to reversed-phase chromatography column ODS-80Ts (column volumeof 1.66 cc, resin particle diameter 20 μm, TOSOH Co., Ltd.) previouslyequilibrated with 1.0% acetonitrile containing 1% acetic acid. After thecolumn was washed with the equilibration solution in a volume of 5columns, [Lys (Boc)^(11, 16, 19, 20, 24)]-rGhrelin (6-28) was eluted bya linear gradient of from 1.0% acetonitrile to 90.0% acetonitrile eachcontaining 1% acetic acid, which was programmed to be finished in avolume of 5 columns. Main fractions were lyophilized to give 6.2 mg ofthe desired protected peptide.

Fragment condensation and de-protection

Triethylamine (51.0 μl, 0.366 mmol) and a solution of di-t-butylbicarbonate (78.0 mg, 0.0356 mmol) in TFE (4.00 ml) were addedrespectively to a solution of Ghrelin (1-5) (190 mg, 0.0301 mmol,Compound 31) in trifluoroethanol (TFE) (6.00 ml) and stirred at roomtemperature for 13 hours. The solvent was evaporated, and ether (20.0ml) was added to the resulting residues, whereby 180.5 mg(N^(α)-Boc)-rGhrelin (1-5) was obtained.

Then, HOSu (5.20 mg, 0.0452 mmol) was added to a solution of[N^(α)-Boc]-rGhrelin (1-5) (22.0 mg, 0.0301 mmol) in DMF (1.00 ml), andDIPCI (7.30 μl, 0.0466 mmol) was added thereto in a bath at −30° C.After the mixture was stirred in the bath at −30° C. for 1 hour and thenat room temperature for 18 hours, the solvent was evaporated, and theresulting residues were converted into powder with ether to give 14.1 mg[N^(α)-Boc]-rGhrelin (1-5)-OSu as a succinimide ester of[N^(α)-Boc]-rGhrelin (1-5).

Then, [N^(α)-Boc]-rGhrelin (1-5)-OSu (3.3 mg, 3.96 μmol) andtriethylamine (2.5 μl, 17.9 μmol) were added to a solution in DMF (0.6ml) of [Lys(Boc)^(11, 16, 19, 20, 24)]-rGhrelin (6-28) (6.10 mg, 2.18μmol) prepared by the recombinant method and stirred at room temperaturefor 24 hours. The solvent was evaporated, and TFA (2.00 ml) was addeddirectly to the resulting residues under cooling on ice and stirred atroom temperature for 1.5 hours. The TFA was evaporated, and ether wasadded to the residues, whereby 6.2 mg crude peptide containing Ghrelin(1-28) was obtained.

This product was dissolved in 2 ml of 5% acetic acid (AcOH) and appliedto YMC-Pack-ODS-A (5 μm, 20 mm×250 mm) and eluted by a linear gradient(flow rate: 10 ml/min.) of from 0 to 95% acetonitrile in 0.1%trifluoroacetic acid for 60 minutes. The desired fractions werecollected, lyophilized, applied to YMC-Pack PROTEIN-RP (C4, 10 mm×250mm) and eluted by a linear gradient (flow rate: 4.7 ml/min.) of from 7.5to 21.3% acetonitrile in 0.1% trifluoroacetic acid for 30 minutes.

The desired fractions were collected, lyophilized and applied toYMC-Pack PROTEIN-RP (C4, 10 mm×250 mm) and eluted by a linear gradient(flow rate: 4.7 ml/min.) of from 7.5 to 21.3% acetonitrile in 0.1%trifluoroacetic acid for 30 minutes. The desired fractions werecollected and lyophilized to give 2.1 mg rGhrelin (1-28). This productshowed a retention time agreeing with that of standard rGhrelin (1-28)in analytical HPLC, and had an intracellular Ca-releasing activity ofEC₅₀=1.5 nM which was equivalent to natural ghrelin.

ESI-MS 3315.0 (theoretical: 3314.8), amino acid composition: Ser; 3.74(4), Glx; 5.69 (6), Gly; 1.18 (1), Ala; 2.05 (2), Leu; 2, Phe; 0.98 (1),Lys; 4.98 (5), His; 1.03 (1), Arg; 1.96 (2), Pro; 4.01 (4)

Compound 87 [^(D)leu⁵]-rGhrelin (1-28)

As a by product in succinimide esterification of [N^(α)-Boc]-rGhrelin(1-5) or condensation of the fragments, 0.8 mg [^(D)leu⁵]-rGhrelin(1-28) was obtained. Its intracellular Ca-releasing activity wasEC₅₀=220 nM.

ESI-MS 3315.0 (theoretical: 3314.8), amino acid composition: Ser; 3.80(4), Glx; 5.92 (6), Gly; 1.23 (1), Ala; 2.07 (2), Leu; 2, Phe; 0.97 (1),Lys; 4.92 (5), His; 1.02 (1), Arg; 1.97 (2), Pro; 4.11 (4)

GC-MS analysis of leucine after hydrolysis in D₂O/DCl: L-Leu; 1.17 (1),D-Leu; 0.83 (1)

INDUSTRIAL APPLICABILITY

By administering the new peptide-type compound of the present inventionor a pharmaceutically acceptable salt thereof into humans or animals, itdemonstrates an excellent working effect as a pharmaceutical preparationfor promoting growth of children and ameliorating the defect ofmetabolic functions caused by GH deficiency, by inducing GH secretionwithout causing substantial side effects, and its antibody demonstratesan excellent working effect as an agent for diagnosis of diseasesattributable to GH deficiency and as a research tool in the field ofscience.

1. A peptide-type compound, wherein in a peptide having the activity ofincreasing the intracellular calcium ion concentration, at least oneamino acid is replaced by a modified amino acid and/or a non-amino acidcompound, or a pharmaceutically acceptable salt thereof. 2-36.(canceled)
 37. A method for treatment of diseases attributable to adefect or decrease in growth hormone, which comprises administering apharmaceutical composition comprising the peptide-type compound claim 1or a pharmaceutically acceptable salt thereof as an active ingredient.38. A method for treatment of diseases not attributable to a defect ordecrease in growth hormone, which comprises administering an agent fortreating diseases not attributable to a defect or decrease in growthhormone and the peptide-type compound of claim 1 or a pharmaceuticallyacceptable salt thereof.
 39. The treatment method according to claim 37,which is applied to animals other than human beings.
 40. A DNA codingfor an amino acid sequence of the peptide-type compound of claim 1,which comprises a nucleotide sequence coding for a peptide containing anamino acid sequence recognizing at least one modifiable amino acid inthe amino acid sequence encoded by said DNA.
 41. The DNA according toclaim 40, wherein the nucleotide sequence is one nucleotide sequenceselected from the group consisting of nucleotide sequences set forth inSEQ ID NOS: 6, 7, 14, 15, 20, 21, 24, 36, 37, 38 and
 39. 42. The DNAaccording to claim 40, wherein the nucleotide sequence is an aminoacid-coding nucleotide sequence in one nucleotide sequence selected fromthe group consisting of nucleotide sequences set forth in SEQ ID NOS: 6,7, 14, 15, 20, 21, 24, 36, 37, 38 and
 39. 43. A vector comprising theDNA of claim
 40. 44. Cells comprising the vector described in claim 43.45. Cells comprising the DNA described in claim 40, wherein apeptide-type compound having an amino acid sequence encoded by said DNAcan be produced as a peptide-type compound having at least one aminoacid modified in said amino acid sequence. 46-48. (canceled)
 49. Amethod for producing the peptide-type compound described in claim 1 bygenetic recombination technology, which comprises transforming a vectorcontaining a DNA coding for an amino acid sequence of the peptide-typecompound, which comprises a nucleotide sequence coding for a peptidecontaining an amino acid sequence recognizing at least one modifiableamino acid in the amino acid sequence encoded by said DNA, into hostcells capable of modifying a side chain of at least one amino acid insaid peptide, then culturing the resulting transformed cells andrecovering the desired peptide-type compound from the culture.
 50. Amethod for producing the peptide-type compound described in claim 1 bygenetic recombination technology, which comprises transforming a vectorcontaining a DNA coding for an amino acid sequence of the peptide-typecompound, which comprises a nucleotide sequence coding for a peptidecontaining an amino acid sequence recognizing at least one modifiableamino acid in the amino acid sequence encoded by said DNA, into hostcells, then culturing the resulting transformed cells and recovering thedesired peptide-type compound from the culture, followed by chemicallymodifying an arbitrary amino acid thereof.
 51. A method for producingthe peptide-type compound of claim 1 further comprising a modified aminoacid with a fatty acid bound via an ester linkage to a side-chainhydroxyl group of the modified amino acid by genetic recombinationtechnology, which comprises using cells having the activity of binding afatty acid via an ester linkage to a side-chain hydroxyl group of anamino acid or via a thioester linkage to a side-chain mercapto group ofan amino acid in the peptide-type compound.
 52. A method for producingthe peptide-type compound of claim 1 further comprising a modified aminoacid with a fatty acid bound via an ester linkage to a side-chainhydroxyl group of the modified amino acid, which comprises using cellshaving the serine acylation activity of binding a fatty acid via anester linkage to a side-chain hydroxyl group of serine in the amino acidsequence set forth in SEQ ID NO:
 8. 53. A method for producing thepeptide-type compound of claim 1 further comprising a modified aminoacid with a fatty acid bound via an ester linkage to a side-chainhydroxyl group of the modified amino acid, which comprises using cellshaving the acylation activity of binding a fatty acid via an esterlinkage to a side-chain hydroxyl group of threonine in the amino acidsequence set forth in SEQ ID NO:
 28. 54. A pharmaceutical compositionfor gene therapy for treatment of diseases attributable to a defect ordecrease in growth hormone, which comprises integrating a vectorcontaining a DNA coding for an amino acid sequence of a peptide-typecompound described in claim 1 into cells in a living body and expressinga peptide with at least one modified amino acid, the peptide having theactivity of increasing the intracellular calcium ion concentration. 55.(canceled)
 56. A pharmaceutical composition for gene therapy fortreatment of diseases not attributable to a defect or decrease in growthhormone, which comprises integrating a vector containing a DNA codingfor an amino acid sequence of a peptide-type compound described in claim1 into cells in a living body and expressing a peptide with at least onemodified amino acid, the peptide having the activity of increasing theintracellular calcium ion concentration.
 57. (canceled)